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1991. 60:513-52
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EUKi\RYOTIC DNA POLYMERASES Teresa S.-F. Wang Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 KEY WORDS :
DN A replication proteins, D NA replication, cell growth control, cell cycle, gene expression.
CONTENTS PERSPEC TIVES . . . . . . . . . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . NOMENCL ATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PROPER TIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein Structures and Enzymatic Prop erties . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catalytic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ASSOCI ATED AC TIVI TIES AND E FFEC TORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Primase .............................................................................................
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3'-5'Exonuclease ................................................................................. Proliferatin g Cell Nuclear Antigen (PCNA ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . R eplication Factor A (RF-A ) .. .............................................. ............. ...... R eplication Factor C (RF·C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T Alltigen...........................................................................................
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523 524 525
GE NETIC STR UCTURE AND PRE DICTE D FUNCTIO NAL DOM AI NS . . . . . . . . . . . . . . . Chromosome Localization....................................................................... Conservation of Primary Sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ ....... Functional Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
527 527
GENE EXP RESSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Tran�formed Cells and Sp ecific Cell Typ es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . During Activation of Quiescent Cell to Proliferate........................................ In Termina l Dijferelltiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . In the Cell Cycle. . . . . . . . . . . . . . ............................................. .............. ......... Cis-Acting Elements in the Upstream Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PHYSIOLOGIC AL ROLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R eplicative DNA Polym erases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles in R eplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles in DNA Repair Synthesis................................................................ CONCL USIO NS AND PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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PERSPECTIVES Understanding the molecular mechanisms regulating eukaryotic cell growth and division is a major endeavor in biological science. To achieve this goal it is essential to comprehend two related but separate biological problems. First, what molecular mechanisms determine whether cells should be maintained in quiescence, allowed to temlinally differentiate, or actively proliferate? Second, in actively proliferating cells, what mechanisms regulate the progres sion through stages of the cell cycle? DNA replication is essential not only for duplication of the genome but also for maintenance of genome integrity during DNA repair. Understanding the mechanisms that ensure that DNA replication does not occur in nonproliferating cells, and those that ensure that each chromosome is replicated only once during the S phase of the cell cycle, are major goals of biological science. A prerequisite to understanding the molecular mechanisms of DNA replication is a comprehension of the proteins involved in this process. Among the essential proteins for DNA replication are the DNA polymerases. Progress in studying DNA polymerases from eukaryotic cells has been problematic due to the low abundance of DNA polymerases in cells, pro teolysis during purification, and the lack of amenable genetics. Much pro gress has been made in the past few years, predominantly by the development of a cell-free reconstituted Simian virus (SV40) DNA replication system ( l , 2), and the isolation and identification o f genes and cDNAs o f DNA polymerases from Saccharomyces cerevisiae (hereafter, budding yeast) and from mammalian cells (3-8). The cell-free reconstitution of SV40 viral DNA replication provides a system for identifying polymerases that are essential for SV40 viral DNA replication and most likely function similarly in host mammalian cells. Three types of eukaryotic DNA-polymerizing enzymes have been described to date. One type of enzyme, named DNA polymerase, polymerizes DNA utilizing DNA as template; a second type, named reverse transcriptase, synthesizes DNA utilizing RNA as template; and a third type, named terminal transferase, polymerizes on the 3'OH-terminus of DNA independent of template. Five eukaryotic cellular DNA template-dependent DNA polymerases exist, designated as DNA polymerase a, {3, -y, 8, and € and encoded by distinct genes. Several recent reviews have discussed the roles of polymerases involved in cell-free replication of viral DNA (1, 2, 9, 10). Other reviews have described polymerase protein structure and en zymology in budding yeast and other higher eukaryotic cells (9, 11-13). This review focuses on the five cellular eukaryotic DNA polymerases. I discuss their properties, their associated activities and effectors, their genetic loci, their genetic structures, the conservation of their genetic and protein sequences, their functional domains, and the regulation of their
EUKARYOTIC DNA POLYMERASES
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gene expression during cell growth and through the cell cycle. The func tional role of each DNA polymerase in replication and repair synthesis is also discussed.
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NOMENCLATURE The recent identification of genes from budding yeast and mammalian cells has defined five distinct species of cellular DNA polymerases (3-8 , 14, 15). DNA polymerase a, a relatively high-molecular-weight enzyme, was the first eukaryotic DNA polymerase identified ( 1 6) . A low-molecular-weight nuclear DNA polymerase found only in vertebrates and not in budding yeast nor other lower eukaryotes is designated as DNA polymerase {3 (17, 18). The polymerase required for mitochondrial DNA replication but encoded in the nucleus is named DNA polymerase y ( 1 9-24) . This polymerase is the product of the MIl'I gene of budding yeast (15). The catalytic subunits for the three budding yeast DNA polymerases are encoded by the genes POLl, POL2, and POL3 for DNA polymerases I, II, and III, respectively. The previous Roman numeral designation of the three budding yeast DNA polymerases, Poll, PollI, and PolIII, has now been revised to polymerases a, 1:, and 6, respec tively (25). In mammalian cells, two DNA polymerases that contain tightly associated 3 '-5' exonuclease activities but respond differently to proliferating cell nuclear antigen (PCNA) for processive DNA synthesis have been de scribed (26-35). These two forms of polymerase 6 are distinct based on their dependency on PCNA for DNA synthetic processivity, and have therefore been described as PCNA-dependent and PCNA-independent polymerase 6 (33, 34). The latter was also described as DNA polymerase 6II (27, 36, 37). The PCNA-dependent polymerase 8 has now formally been designated as DNA polymerase 8, while the polymerase 811 or PCNA-independent polymerase 6 (homolog of yeast polymerase II) has now been named DNA polymeras\; E (25, 33). Much confusion was created in the literature in the past few years by naming exonuclease-associated DNA polymerases "polymerase 8." Two nuclease-associated DNA polymerase activities, one from bone marrow (38) and another 170-kDa exonuclease-associated polymerase purified from human placenta, were both described as polymerase 6 (39). However, their dependency on PCNA for DNA synthetic processivity is still unknown , and this criterion cannot be used to classify these two polymerases as either polymerase 6 or polymerase E. Another polymerase activity from calf thymus with a readily dissociable nuclease and an associ ated primase activity was described as DNA polymerase 61 ( 27, 36). It has been suggested that this represents an enzyme fraction containing polymerase a and a 3' -5' exonuclease (27, 40).
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PROPERTIES
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Protein Structures and Enzymatic Properties PROTEIN STRUCTURE The protein structure of DNA polymerase a was a controversial issue for decades. Improvement of the conventional biochemical purification procedure and development of the imunnoaffinity purification method have facilitated the resolution of the structure of this enzyme. The subunit components of DNA polymerase a isolated from a wide range of phylogenetic species either by immunoaffinity purification protocol or by conventional chromatographic methods contain a remarkably similar set of four polypeptides (12, 4 1 56) Polymerase a is composed of (a) a cluster of related high-molecular-weight polypeptides predominantly of 165- 1 80 kDa containing the catalytic function that are derivatives of a single primary polypeptide (44, 45); (b) a polypeptide of about 70 kDa with unknown catalytic function; and (c) two polypeptides of 55-60 kDa and 48-49 kDa associated with primase activity (reviewed in 57, 43, 46-48, 50, 58-70). Thus an appropriate designation for this four-subunit enzyme complex is DNA polymerase a/primase ( 1 1, 1 2) . The isolation of polymerase a full length complementary DNA (eDNA) from human cells and the gene from budding yeast defines the catalytic polypeptide as a 1 65-kDa protein (4, 5). The often observed cluster of polypeptides with a molecular mass higher than the deduced primary sequence results from posttranslational modification, while the often observed lower-molecular-mass polypeptides ranging from 1 20 to 1 65 kDa result from proteolysis of the posttranslationally modified protein at specific l abile site(s) within the N-terminus (7 1 ) . DNA polymerase f3 isolated from all vertebrate species i s a 40-kDa protein ( 1 8, 72-78). Isolation of the DNA polymerase f3 gene from rodent and human cells confirms the structure of this enzyme as a 335-amino-acid protein with a molecular size of 39 kDa ( 14, 79, 80) . A functional homolog has not yet been found in budding yeast. The protein structure of DNA polymerase l' has also been controversial due to its low abundance and proteolytic degradation during purification. Purifica tion of DNA polymerase l' from chick embryo suggested that the enzyme is a homotetramer of 47-kDa subunits (8 1 , 82) . This enzyme purified from Dro sophila melanogaster is composed of a 1 25-kDa catalytic polypeptide and an additional subunit of 35 kDa in a I : 1 ratio (23). Polymerase l' purified from Xenopus laevis contains a single polypeptide of 140 kDa reported to be associated with catalytic function (24). The confirmation that MIPI from budding yeast is the gene encoding the yeast mitochondrial DNA replication enzyme, polymerase 1', clearly defines the structure of this polymerase as a protein of 143 .5 kDa ( 15). Identification of the budding yeast POL3 (CDC2) gene encoding DNA -
.
EUKARYOTIC DNA POLYMERASES
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polymerasf� () defines the molecular mass of the catalytic subunit of this polymerasf� as 125 kDa (6). B iochemical purification of polymerase () activity
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from calf thymus and HeLa cells demonstrates an enzyme composed of a 125-kDa catalytic polypeptide and an associated 48-kDa subunit of unknown function (29, 33). Polymerase E purified from HeLa cells was reported to be a protein of 2 15
kDa (32, 33) . DNA polymerase E (polymerase II) from budding yeast was described as a group of proteins ranging in molecular size from 132 to 200 kDa containing polymerase activity (83, 84). The recently isolated gene for polymerast: E from budding yeast encodes a protein of 255 kDa that is by far the largest DNA polymerase yet described (8). It is assumed that the pre viously observed lower-molecular-mass proteins from either mammalian cells or yeast were proteolytically degraded products of the 255-kDa polypeptide. Two polypeptides of 80 and 34 kDa from yeast polymerase E enzyme prepara
tions have been provisionally assigned as subunit components of this enzyme (A. Sugino, personal communication). In contrast, polymerase E purified from HeLa cells does not contain these subunits (32, 33) . The general enzymatic properties such as primer template preference, divalent cation preferences, salt sensitivity, and in hibitors of these five distinct polymerases have been described by others (13, 18, 29, 83, 85). Properties that can be used to distinguish these DNA ENZYMATIC PROPERTIES
polymerases are summarized in Table 1 and are not discussed here in detail. Two notable enzymatic properties of DNA polymerases are processivity and fidelity. Processivity of a polymerase is the extent of DNA chain el onga tion by DNA polymerase per interaction with the 3' OH-end of the DNA
primer. Processivity of DNA polymerases a and f3 has been studied ex tensively. For DNA polymerase a, depending on the assay conditions , purity of the enzyme, integrity of the catalytic polypeptide, and the subunit com ponents of the enzyme fraction used, a large variation in the number of nucleotides. polymerized per binding event has been reported (86-88). Com pared to the other four eukaryotic DNA polymerases, polymerase a is gener ally regarded as moderately processive (Table l ) . Despite the dependency of
processivity on reaction conditions and polymerase a integrity, processivity has been used as a parameter to identify a variety of effectors and accessory proteins for polymerase a, which have been reported to enhance its processiv ity (89 -92). With the exception of an accessory protein RF-A, the in vivo significance of these identified effectors is not yet clear. DNA polymerase f3 is the smallest DNA polymera se in eukaryot ic cel ls,
but it has several unique and distinct enzymatic properties. It is insensitive to aphidicolin, sulfhydryl reagents, salt, and butylphenyl-dGTP ( 18, 93), but is sensitive to ddNTPs (18, 86). Processivity of polymerase f3 depends on the
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Table 1
Eukaryotic DNA polymerases: protein structure, cellular location, and properties DNA polymerase
{3
'Y
8
165"
40
140
125
255
70 58 48
none
unknown
48
unknown
Protein structure catalytic polypeptide (kDa) associated subunits
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(kDa)
Yeast gene
POLl none (CDC] 7)
MIPl
POLJ (CDC2)
POL2
Cellular location
nuclear
nuclear
mito
nuclear
nuclear
Associated enzyme activities
3' -5' exonuclease
noneb
none
yes
yes
yes
primase
yes
none
none
none
none
med
low
high
low
high
Properties processivity inherent
high
with PCNA fidelity
high
low
high
high
high
yes
Inhibitors aphidicolin
yes
no
no
yes
N-ethylmaleimide
yes
no
yes
yes
unknown
butylphenyl-dGTP
yes
no
no
no
no
dideoxynuc1eoside 5' -triphosphate
no
yes
med
no
no
no
unknown
unknown
yes
med
(ddNTP) carbonyldiphosphonate
Molecular mass deduced from primary protein sequence. b Cryptic exonuclease in the isolated catalytic polypeptide was reported in Drosophila melanogaster embryo
a
(102).
metal ion in the reaction; it is five times higher in reactions with Mn2+ than in reactions with Mg2+ as activator
(94).
Polymerase 'Y is aphidicolin resistant but N-ethylmaleimide sensitive and is moderately inhibited by dideoxythymidine triphosphate (ddTTP) merase 'Y has been reported to be highly processive Polymerases
{) and
E
(24). Poly in DNA synthesis (18).
are distinguished from the other DNA polymerases by
their readily detectable proofreading 3' -5' exonuclease activity. Both are aphidicolin sensitive
(29, 32, 33, 36), and both are rather insensitive to the
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EUKARYOTIC DNA POLYMERASES
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inhibition of butylphenyl-dGTP. Furthermore, while polymerase 8 is stimu lated by 10% dimethyl sulfoxide (DMSO), polymerase € is inhibited by this reagent (33). These two DNA polymerases are immunologically and structur ally distinct from polymerase a, but have some immunological and structural similarity with each other (34) . Interestingly, these two polymerases are strikingly distinct in their processivity. Polymerase 8 has low processivity with long single-stranded templates, but, in the presence of peNA, polymerase 0 can perform highly processive DNA synthesis on this template. Thus, PCNA has been designated as the auxiliary protein of DNA polymerase 8 (95). In contrast to polymerase 8, DNA polymerase E has an inherently high processivity on this template in the absence of peNA in reactions with low concentrations of Mg 2+ (30, 32, 3 3 , 84) . DNA synthetic fidelity and mutation specificity of polymerases a, {3, and 'Y have been studied extensively (reviewed in 1 8 , 96-99). A detailed discussion of fidelity studies and the mutation specificity of each polymerase is beyond the scope of this review. A summary of these studies is presented in Table 1. Several interesting findings are noteworthy. Despite the lack of a measurable proofreading 3 ' -5 ' exonuclease in polymerase a/primase complex , the mis incorporation rate of this enzyme is low. Two factors are suggested to influence the fidelity of polymerase a. First, the more intact form of the four-subunit complex of DNA polymerase l1'/primase has higher DNA syn thetic accuracy than aged and proteolyzed enzyme ( 1 00, 1 0 1 ). A second factor is the presence of a proofreading exonuclease activity. A cryptic 3' -5' exonuclease activity in the large catalytic subunit of polymerase a from D . melanogaster embryo has been described when this subunit i s separated from the 70-kDa subunit ( 1 02). This unmasked cryptic exonuclease in the catalytic polypeptide enhances the fidelity of DNA synthesis 1 00-fold. Furthermore, this separated polymerase a catalytic polypeptide with the cryptic exonucle ase has altered sensitivity to inhibitors. Like polymerases 8 and E, it hecomes less sensitive to butylphenyl-dGTP , whereas this analog is a potent inhibitor of the intact polymerase a/primase complex ( 1 03) . Moreover, a polymerase a enzyme preparation from human lymphocyte containing a loosely associated proofreading exonuclease was reported to have higher DNA synthetic fidelity ( 1 04) . Consistent with the notion that proofreading exonuclease enhances polymerase DNA synthetic fidelity, the fidelity of calf thymus DNA polymerase E (previously described as polymerase 811) has an accuracy 1 0and 500-fold greater than that of polymerases a and {3, respectively (37).
Catalytic Mechanisms Using steady-state kinetics analysis and supplemented by direct physical measurement, the catalytic mechanisms of DNA polymerases a and {3 were
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thoroughly investigated (reviewed in 105 and 45,86,94, 105-Ill). Exten sive studies of DNA polymerase a and substrate interaction were performed
with the biochemically purified but proteolyzed form of the enzyme ( 1 1 2) . Most o f these polymerase a and substrate interaction mechanisms were later
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proven to be qualitatively identical when reassessed with the immunopurified polymerase a/primase complex (44) . In a separate study, analysis with im munoaffinity-purified calf thymus DNA polymerase a demonstrated quali tatively higher affinity for deoxynucleoside 5' -triphosphates (dNTPs) and
primer-template (52). These studies suggest that the intact form of DNA polymerase
a
might
yield quantitatively more efficient enzyme for the assessment of catalytic mechanism. The interaction of polymerase a and substrates was found to obey a rigidly ordered sequential ter mechanism. The order of the sequential ler mechanism is first, interaction with template (single-stranded DNA),
second, interaction with primer stem, and third, interaction with dNTP. Specification of the addition of dNTP to the enzyme is determined by the template sequence. A minimum length of 8 nucleotides of primer is required for effective binding with polymerase a. The terminal 3-5 nucleotides of the primer must be template-complementary . A single mispaired terminal nucleo tide prevents correct primer binding and the subsequent step of dNTP addition to the enzyme ( 106- 1 1 0). This finding was later confirmed by assessment of rate of extension from mispaired primers by calf thymus polymerase a ( 1 1 3 , 1 14). The rate o f extension from mispairs is 103 to 106 times slower than from a paired primer terminus ( 1 14). Hydrolysis of the mispaired primer residue by either the f subunit of Escherichia coli polymerase III or the proofreading 3' exonuclease associated in polymerase {) permits subsequent extension and increases the fidelity of DNA polymerase a ( 1 1 5 , Il6). 3'-Terminal H or OH, but not P04, can interact with polymerase a. The primer binding occurs through the coordinated participation of four Mg 2+ -primer binding subsites, which may serve as a shuttle to facilitate not only phosphodiester bond formation but also polymerase translocation. The catalytically active
polymerase
a
appears to have two positively cooperative single-stranded
DNA-binding sites. Thus, it was proposed that polymerase a is a con formationally active enzyme that responds to signals from template sequence that are transduced via template-binding site(s) interaction ( 1 09, 1 10). Unlike polymerase a, DNA polymerase {3 has a weak affinity for single stranded DNA that is not modulated by base sequence and not enhanced by the presence of potentially base-paired 3' -OH termini. Polymerase f3 has no affinity for intact duplex DNA, but has a high affin ity for nicked duplex DNA with nicked termini either bearing 3' -OH or 3' -P04 residues. Polymerase f3 can perform a limited strand displacement synthesis at the 3 ' -OH termini of the nicked DNA ( 1 1 1). In corroboration of these earlier findings , polymerase
EUKARYOTIC DNA POLYMERASES
521
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{3 from HeLa cells was also demonstrated to perform limited strand displace ment synthesis on 3' -OH termini in DNA produced by AP endonuclease at the 5' side of apurinic sites. Polymerase {3 is able to insert a single com plementary nucleotide into the single nucleotide gap created (117).
Polymerase {3 purified from human cells has less stringent requirements for primer interaction than polymerase a. Polymerase {3 is reactive with primers that contain 1-3 mispaired terminal residues. Polymerase {3 purified from mouse cells was reported to carry out a two substrate-two product reaction that follows ordered bi-bi kinetics: interacting with primer template first, followed by dNTP ( 1 1 8) . The mechanism of substrate interaction of human polymerast! f3 is also rigidly ordered, and sequential by binding to DNA first followed by dNTP. However, binding to primer-template appears to occur by a concerted mechanism. The primary signal for catalytic productive interac tion of polymerase {3 with DNA is a base-paired primer moiety that must be adjacent to a short length of potentially single-stranded template. The mini mum length of this template and processivity are influenced by the divalent cation. With Mg2+ , minimum template length is >5 nuc1eotides and polymerization is distributive, whereas, with Mn2+ , the minimum required template is a single nucleotide and the polymerization is more processive by inserting 4-6 residues per cycle of primer interaction (94, Il l) . Consistent with the notion that higher processivity is responsible for higher fidelity, the error rate at hot spots for polymerase {3 was also found to be lower in reactions with Mn2+ as cation ( 1 19) . ASSOCIATED ACTIVITIES AND EFFECTORS
Primase All DNA polymerases require a 3' -hydroxyl terminus of a preexisting primer for reaction ( 120, 121). DNA polymerase a is the only eukaryotic polymerase with a tightly associated primase. Primase activity resides in a heterodimer of 55-60 kDa and 48--49 kDa polypeptides that have been purified from various biological systems as the associated activity and subunit component of polymerase a. The purification, enzymatic properties, and reaction mech anisms of primase reported from numerous laboratories have been reviewed (57). Some: recent developments that were not included in the previous review are discussed here. The genes for both primase subunits from budding yeast are now cloned and designated as PRJ1 and PR12. By gene disruption, both genes were found to be essential (69, 122, 123). By affinity labeling, the ATP- or GTP-binding site(s) of primase from calf thymus and yeast were found to be localized exclusively in the 48-kDa subunit (67 , 124). A monoclonal antibody against
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the 60-kDa subunit of chick primase has been produced. Immunofluorescence demonstrates the exclusive nuclear localization of primase in granular struc tures bound to the nuclear matrix. Furthermore, primase antigen was reduced in quiescent cells and induced when quiescent cells were activated to pro liferate by serum addition. These results suggest that the expression and organization of primase in the granular structures on the nuclear matrix are regulated in correlation with cell proliferation (125). The cDNA of the 49-kDa subunit of mouse primase was cloned and characterized (126). Se quence comparison with yeast primase 49-kDa subunit shows a high con servation of amino acid sequences in the N-terminal halves of the polypeptides and a potential metal-binding domain (Zn finger) in this region. The C-terminal region diverges from yeast primase. During serum induction of quiescent cells to proliferate, expression of the mouse 49-kDa primase gene lO-fold at the steady state transcript level, but in is positively induced actively proliferating cells the gene is constitutively expressed throughout the cell cycle (127). This finding is in agreement with the immunofluorescence data reported for chick primase (125). The cDNA for the 59-kDa large subunit of mouse primase has also been cloned (B. Y. Tseng, personal communica tion). �
3' -5' Exonuclease None of the five cellular DNA polymerases has an associated endonuclease or 5'-3' exonuclease. Polymerases y, S, and € do have an associated proofread ing 3'-5' exonuclease. The proofreading 3'-5' exonuclease and polymerase y activity have been suggested to reside in the same polypeptide. From X. iaevis, the 3' -5' exonuclease activity and polymerase y activity copurified in a constant ratio in the final three steps of purification. The two enzymatic activities also cosediment in a glycerol gradient under partially denaturing conditions (128). In chick embryo, the 3' -5' exonuclease was found to coprecipitate with DNA polymerase y during immunoprecipitation with anti DNA polymerase yantibody (129). Like the classic proofreading nuclease of E. coli polymerase I and T4 polymerase (130, 131), the 3'-5' exonuclease of polymerase y hydrolyzes mismatched termini with significantly greater effi ciency than base-paired termini (128, 129). The 3' -5' exonuclease activities in polymerases {) and E also behave as typical proofreading nucleases with the preference for hydrolyzing mismatched over base-paired termini (32, 83, 84, 132). As with the proofreading 3' -5' exonuclease in prokaryotes, budding yeast polymerase 8 also has a demonstrable turnover of dTTP to dTMP by the successive actions of polymerase and exonuclease (132). Based on the in hibitor studies and the constant ratio of nuclease to polymerase activity in the final steps of purification, it is suggested that the 3' -5' exonuclease activity of both polymerases S and € reside in the same polypeptide as the polymerase
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EUKARYOTIC DNA POLYMERASES
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activity (26, 38 , 83, 84) . As described above, a cryptic 3' -5' exonuclease was also reported in the catalytic polypeptide of Drosophila polymerase a when isolated from the other subunits ( 1 02). An 3' -5' exonuclease activity has been described jin recombinant yeast polymerase a 1 80-kDa subunit recently ( 1 32a) . This 3'-5' exonuclease activity is not a conventional proofreading nuclease, however. The yeast 1 80-kDa subunit, as well as the 1 80-kDa/86kDa complex and the four-subunit polymerase a/primase complex, was cap able of removing 3' terminal nucleotide of poly(dT)6oo- 32p-dNMPo.4-0.5' The physiological significance of this yeast 1 80-kDa associated exonuclease is not yet known. The catalytic polypeptide of human DNA polymerase a has been overproduced from recombinant baculovirus-infected insect cells as a single polypeptide of 1 80 kDa. This single-polypeptide polymerase a does not have a detectable proofreading 3' -5' exonuclease activity when assayed with either mismatched or matched base-paired termini (W. Copeland and T. Wang, manuscript submitted). It is interesting that the cryptic proofreading exonucle ase of DNA polymerase a appears to be unique for the embryo of Drosophila.
Proliferating Cell Nuclear Antigen (PCNA) An auxiliary protein for DNA polymerase 8 that enhances its processivity was found to be immunologically , structurally , and functionally identical with proliferating cell nuclear antigen, PCNA (29, 95, 133, 133a, 1 34). PCNA is a 36-kDa nuclear protein reactive with sera from a subset of patients with the autoimmune disease systemic lupus erythematosus (SLE), and this protein was only found in actively proliferating cells ( 1 35 , 1 36) . PCNA was also found to be essential for in vitro SV40 DNA replication (133, 1 37). Reviews of the role of PCNA in viral and cellular DNA replication and cell growth have been published recently (2, 1 3, 1 38). The gene and cDNA for PCNA have been isolated and characterized from rat , human, budding yeast, and Drosophila. Protein sequence comparison indicates that PCNA is a highly conserved protein among these eukaryotes ( 1 39-1 43) . Budding yeast PCNA gene (POL30), like the budding yeast polymerase 8, is periodically expressed at the level of steady-state mRNA in starve-feed cultures . During meiosis, the mRNA level also increases prior to initiation of premeiotic DNA synthesis ( 14 1 ) . In contrast to budding yeast, the protein level of PCNA in mammalian cells was reported to be constitutively expressed throughout the cell cycle, although PCNA mRNA and protein levels were dramatically increased during activation of quiescent cells to proliferate ( 1 40, 1 44). PCNA does not appear to have a stringent species specificity for i ts interaction with polymerase 8 and other replication factors . Calf PCNA can stimulate yeast polymerase 8 ( 1 45); calf thymus PCNA and human RF-C (a replication factor, see discussion below) are able to stimulate budding yeast
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polymerase S DNA synthesis on singly primed M 1 3 single-stranded DNA in an ATP-dependent manner ( 1 46). Drosophila PCNA could substitute for human PCNA, although with reduced efficiency, in stimulating SV40 in vitro DNA replication ( 143). PCNA has also been shown to affect the DNA synthetic activity of polymerase E. Under certain salt conditions in the pres ence of RF-C, calf thymus PCNA can enhance budding yeast polymerase E DNA synthetic activity on singly primed single-stranded DNA and can en hance its synthetic processivity in homopolymer reactions (84, 1 46) . In SV40 origin-dependent DNA replication, the absence of PCNA yields short and lagging-strand-specific products, which are predominantly derived from re gions around the SV40 origin ( 1 37). PCNA also stimulates the ATPase activity of RF-C and is an essential component of the primer recognition complex, PCNA/RF-ClATP (92) . Evidence from product analysis of the SV40 in vitro replication reactions suggests that PCNA is the functional equivalent of T4 phage gene 45 protein or E. coli DNA polymerase III f3 subunit ( 1 47). The functional analogy between PCNA and T4 phage gene 45 protein is also reflected in the primary amino acid sequences of these proteins. These two proteins have a 3 1-50% primary sequence similarity within a limited region, but have no significant similarity to dnaN gene product, which encodes the f3 subunit of E. coli DNA polymerase III holoenzyme ( 1 47) . In sum, several lines o f evidence support the conclusion that PCNA does not directly affect the DNA synthetic activity of DNA polymerase a. For polymerase S, synergistic interaction of PCNA with RF-C/ATP and RF-A is required for the optimal DNA synthetic activity on primed single-stranded DNA or in reconstituted SV40 origin containing DNA reaction. Depending on the reaction condition and the presence of RF-C and ATP, the conjecture is that PCNA might also have an effect on in vitro DNA synthetic activity of polymerase E (84, 146).
Replication Factor A (RF-A) RF-A (also named replication protein A, RP-A, and human single-stranded DNA-binding protein, HSSB) is a multisubunit single-stranded DNA-binding protein. RF-A contains three polypeptides of 70, 34, and 1 1 kDa and func tions as an auxiliary protein for both polymerases a and S. RF-A is required for the initiation and elongation stages of in vitro SV40 DNA replication ( 148- 153). Biochemical and immunological evidence indicates that the sing le-stranded DNA-binding activity resides exclusively in the 70-kDa subunit ( 149, 1 5 1 , 1 54) . RF-A alone stimulates DNA polymerase a activity on either primed M 1 3 DNA or oligo dT-primed polydA , resulting in short DNA products; PCNA had no effect on the size of the product. In contrast to the effect on polymerase a, RF-A alone has no specific effect on the reactivity of
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polymerase;; even in the presence of PCNA . However, polymerase;; can be synergistically stimulated by PCNA, RF-A, and RF-C (another replication factor, see discussion below), yielding long DNA products (152-154) . Immunological evidence has suggested that the 70-kDa subunit may be required for DNA unwinding and polymerase S stimulation, whereas both 70and 32-34-kDa subunits have been implicated to be involved in the stimula tion of polymerase a DNA synthetic activity (154). The gene for the 70-kDa subunit has been cloned from budding yeast and found to be essential (155). The deduced protein sequence reveals significant amino acid similarity of this yeast 70-kDa RF-A subunit with its human homolog (155). The cDNA for the 34-kDa subunit of RF-A from human cells was also cloned and overexpressed (156). The 34-kDa protein is a phosphoprotein, and phosphorylation of this subunit is cell cycle regulated with higher phosphorylation occurring at late 01 and early S phase (157) . Yeast RF-A or other single-stranded DNA binding proteins, including E. coli single-stranded binding protein (SS B) and adenovirus and herpes simplex virus DNA-binding proteins, are able to replace human RF-A in the unwinding step in the initial stage of SV40 in vitro DNA replication. However, yeast RF-A and these other SSBs are unable to substitute for human RF-A in the SV40 in vitro DNA replication reaction (1, 2, 153, 158). Thus, like polymerase a, RF-A demonstrates stringent species specificity iln SV40 in vitro DNA replication . It also appears to be involved in genetic recombination (155).
Replication Factor C (RF-C) RF-C (also named activator I, AI) is a multi subunit protein complex that has primer/template binding and DNA-dependent ATPase activity . The intrinsic DNA-dependent ATPase activity of RF-C is stimulated by PCNA (147), and addition of RF-A further stimulates the DNA-dependent ATPase activity (159) . Purified RF-C is composed of subunits of 140, 41, and 37 kDa. The 140-kDa subunit binds primer/template DNA, whereas the 41-kDa subunit binds ATP (92). In SV40 in vitro DNA replication reactions, the absence of RF-C causes accumulation of early replicative intermediates and yields DNA products that hybridize only to the lagging-strand template (92, 152, 160163). This observation suggests that RF-C has a profound effect on leading strand DNA synthesis. RF-C appears to be the functional equivalent of the T4 phage gene 44/62 complex (reviewed in 164 and 165). Similar to the T4 phage DNA replication system, processive DNA synthesis by polymerase 8 on primed M13 single-stranded DNA requires RF-A, RF-C, and PCNA and hydrolysis of ATP or dATP (92, 147, 159, 162, 163, 166) . Furthermore, replication complexes from (a) T4 phage containing gene products 43, 44/62 and 45, or T7 polymerase/thioredoxin complex, (b) E. coli DNA polymerase III holoenzyme, and (c) other DNA polymerases can substitute for the lead-
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ing-strand polymerase complex from human cells in SV40 in vitro DNA replication reaction ( 1 62, 1 68). Extensive product analysis of reactions with RF-C in various combinations with PCNA, ATP, and RF-A, in conjunction with isolation of the replication protein complex by gel filtration, and analysis of the kinetics of protein complex formation, support a model of protein-protein interaction hierarchy at the replication fork. A model has been proposed that binding of RF-C to the lagging-strand polymerase complex at the replication origin occurs first, followed by binding of PCNA in an ATP-dependent manner for the formation of a "primer recognition complex," which in cooperation with RF-A plays a crucial role in initiating the leading-strand synthesis from the nascent DNA fragment synthesized by the lagging-strand polymerase complex (92, 1 62, 1 63 , 1 66). A proposed model showing the effect of RF-A, RF-C, and PCNA on the lagging-strand and leading-strand switching is illustrated in Figure 3 and discussed further in the section of this review on Physiological Roles.
T Antigen In SV40 viral DNA replication, it is reasonable to assume that there are protein-protein interactions between T antigen and cellular replication pro teins during initiation of the SV 40 DNA replication. An earlier report de scribed the association of DNA polymerase a activity from crude cell extracts with T antigen, but purified polymerase a/primase complex failed to demon strate significant interaction with T antigen ( 1 69). These observations suggest that the interaction between T antigen and polymerase a/primase complex might be due to a third yet unidentified cellular protein in the crude extract. A subset of T antigen monoclonal antibodies was shown to prevent the binding of T antigen and polymerase a/primase from crude cell lysate, and in terestingly these antibodies also block T antigen interaction with murine p53 protein (170, 171) . Recently, this issue has been investigated further with the highly purified four-subunit polymerase a/primase complex and T antigen. It has been demonstrated that T antigen binds directly and specifically to the catalytic subunit of human DNA polymerase a. The amino-terminal 83 amino acids of T antigen are both necessary and sufficient for the binding. Further more , polymerase a from human tissue has a lO-fold higher affinity for T antigen than does polymerase a from calf, and conversely human polymerase a that binds the SV40 T antigen with high affinity does not bind polyoma T antigen ( 1 72; I . Domeiter, E. Fanning , personal communication) . This is consistent with an earlier report of species specificity of polymerase a/ primase in SV40 in vitro DNA replication reaction ( 1 73). Thus, T antigen can be classified as another effector for DNA polymerase a in the viral SV40 replication system, perhaps by directing the polymerase to the origin of replication, where T antigen is bound.
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Several other effectors for polymerase 0' have been reported. Among those, two proteins named Cl and C2 can increase the affinity of DNA polymerase 0' to primer stem 20-30-fold. Interestingly, the effect of these two proteins on polymerase 0' also demonstrates species specificity ( 174, 1 75). Another protein, alpha accessory factor (AAF) , is highly specific for polymerase 0' with no effect on polymerases f3, 8, or y. AAF increases the affinity of polymerase a/primase for DNA templates, enhances its processivity, and aIlows this enzyme to traverse double-stranded DNA region ( 1 76, 177). GENETIC STRUCTURE AND PREDICTED FUNCTIONAL DOMAINS
Chromosome Localization The catalytic polypeptide of human DNA polymerase a has been mapped to the short arm of the X-chromosome at Xp2 1 .3 to Xp22. 1 by expression, and later confirmed by genomic Southern hybridization with the cDNA (4, 1 78). The gene for mouse polymerase a catalytic polypeptide was recently localized on the mouse X-chromosome in region C-D ( 1 79). The genes of the two mouse primase p49 and pS8 subunits were localized to mouse chromosomes 10 and 1 , respectively ( 1 79) . The genes coding for all four subunits of budding yeast DNA polymerase 0' were mapped. The catalytic polypeptide maps on yeast chromosome XIV, the 74-86 kDa subunit on chromosome II , and the two primase 58- and 48-kDa subunits on chromosomes XI and IX, respectively ( 1 80) . The chromosome localization of budding yeast polymerase 8 is mapped to the left arm of chromosome IV (6) , whereas the chromosome localization of polymerase E is not yet known. The yeast MIP1 gene encoding mitochondrial DNA polymerase y was mapped to the right arm of yeast chromosome XV ( 1 5). The genes for human and mouse DNA polymerase f3 were both mapped to chromosome 8 of human and mouse, respectively ( 1 8 1- 1 83). Further fine mapping of human and mouse DNA polymerase f3 gene has localized the gene near localization of the tissue plasminogen activator on the proximal short arm of chromosome 8 ( 1 84) .
Conservation of Primary Sequences Sequence comparison of cDNAs and genes of various prokaryotic and eu karyotic DNA polymerases has revealed an evolutionary conservation of three groups of DNA polymerases on the basis of their amino acid sequence relatedness: (a) E. coli DNA polymerase I and phage T7 DNA polymerase ( 1 85); (b) DNA polymerase f3 and terminal transferase (80, 1 86); and (c) a-like DNA pulymerases, including protein-primed replicative DNA polymerases (4, 1 87).
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Comparison of deduced amino acid sequences of DNA polymerase {3 and other proteins showed a sequence similarity with human terminal de oxynucleotidyltransferase (80, 1 86). Sequence alignments revealed conserva tion between residues 42-215 of polymerase f3 and residues 195-366 of terminal transferase. Nearly 30% of these 1 74 amino acid residues of polymerase f3 were identical with those of the 1 72-residue sequence of terminal transferase. Forty residues (23%) represented conservative substitu tion. Thus, the sum of identical residues, the favored substitutions , and the conservative substitutions in amino acid pairs was 1 24 out of 1 74 residues (7 1 . 3%). The overall predicted secondary structures of the two enzymes were also closely similar in their homologous regions (80) . However, the C terminal region (amino acid residues 2 1 6-335) of DNA polymerase f3 has no significant similarity with that of terminal transferase. Interestingly, the similar region is coded exclusively by the third and the later exons. The isolation of the catalytic polypeptide of human DNA polymerase a cDNA and analysis of its sequence revealed striking sequence similarity with replicative DNA polymerases from both prokaryotes and eukaryotes (4, 1 87). Furthermore, by utilizing both immunologic and molecular genetic approaches, the differential evolutionary conservation of four unique epitopes on the catalytic polypeptide and the conservation of genomic DNA sequences have been demonstrated among organisms as phylogenetically disparate as primate and unicellular fungi ( 1 88). These findings have facilitated the identification of many DNA polymerase genes and cDNAs and have led to the recognition of an a-like family of DNA polymerase genes. From amino acid residues 609 to 1085 of human DNA polymerase a , six regions exist that are similar to regions of the three budding yeast DNA polymerases a, 15, and E, a potential budding yeast polymerase gene, REV3, product that is involved in mutagenesis responses, DNA polymerases from the herpes simplex virus (HSY) family, cytomegalovirus (Hey), Epstein-Barr virus (EBV), and vac cinia virus polymerase (4-6, 8 , 45 , 1 87 , 1 89-1 93). Five regions of similarity exist to adenovirus DNA polymerase and bacteriophage T4 DNA polymerase ( 1 94, 1 95). Three regions of similarity exist to DNA polymerases from Bacillus subtilis phage 29 and E. coli phage PRDI ( 1 96, 1 97), as well as to two potential DNA polymerases, yeast linear plasmid pGKLI and maize SI mitochondrial DNA ( 198-200) . The regions described above are designated I to VI according to their extent of similarity, with region I being the most conserved and region VI, the least similar. The significance of these six regions is further underscored by their relative linear spatial arrangements within each polymerase polypeptide (Fig ure 1). The order of these six regions in each polymerase polypeptide is IV -11 -VI-III-I-V, while the distances between each of these conserved regions are variable. While regions I, II, and III appear to be the basic core regions of this a-like DNA polymerase family, yeast MIPI gene encoding the yeast
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polymerase 'Y revealed the presence of regions I, II, and VI but not regions III, IV, and V. Furthermore, the linear spatial order of the three regions, I, II, and VI, is different from that in the other a-like family DNA polymerases (15). Although the sequence of yeast polymerase € demonstrates the presence of these six conserved regions, and the linear spatial arrangements of these regions are similar to those of the other a-like DNA polymerases, the similarity among these six regions is relatively weak. Region I, containing sequence -YGTDTS-, which is the most highly conserved sequence among the a-like family DNA polymerases, is poorly conserved in yeast polymerase € (8). Both yeast DNA polymerases 8 and € also showed distinct sequence similarity to viral polymerases (6, 8). This suggests that these two 3' -5' exonuclease-containing DNA polymerases of yeast might belong to a sub family of a-like polymerases. Recently the dinA gene of E. coli was found to encode the E. coli DNA polymerase II and, interestingly, the sequence of this gene revealls the presence of regions IV, II, III, I, and V sequences of the a-like family polymerase (20 1 ) . The presence of these conserved regions in DNA polyrnerases from human to bacteriophage suggests that this group of DNA polymerases may all have been derived from a common primordial gene. Human DNA pol a
IV
NH2
HH
S. cere'llslae DNA pOlo S. carewlslaa DNA pol £
1
2
MH2
Herpe s Simplel DNA p�1
Cytomegatov!rus DNA POI
Ade novirus
T4 DNA pOI
2
H
N O
MH2
DNApol
I \ 1,
-
_____
NH2 ----
Vaccinia Virus DNA pol
N
pGKl1 S1 Mitochondria
S. cereVlslae A e v 3
"" 2
L..J
H
2
MH2
--- COOH
COOH
/ O--COOH J I \�-.oo "....-... 1 +-.../' r • �---1"-COOH
COOH MH2 +--....+-111-\-. -' COOH \'__j- COOH -+- ....-+-___�1\ -+- ....--t-
Annu. Rev. Biochem. Copyright
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1991. 60:513-52
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Annu. Rev. Biochem. 1991.60:513-552. Downloaded from www.annualreviews.org Access provided by Mahidol University on 01/20/15. For personal use only.
EUKi\RYOTIC DNA POLYMERASES Teresa S.-F. Wang Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 KEY WORDS :
DN A replication proteins, D NA replication, cell growth control, cell cycle, gene expression.
CONTENTS PERSPEC TIVES . . . . . . . . . . . . . . . . .. . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . NOMENCL ATURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PROPER TIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein Structures and Enzymatic Prop erties . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catalytic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ASSOCI ATED AC TIVI TIES AND E FFEC TORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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516 516 519 521
Primase .............................................................................................
521
3'-5'Exonuclease ................................................................................. Proliferatin g Cell Nuclear Antigen (PCNA ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . R eplication Factor A (RF-A ) .. .............................................. ............. ...... R eplication Factor C (RF·C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T Alltigen...........................................................................................
522
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GE NETIC STR UCTURE AND PRE DICTE D FUNCTIO NAL DOM AI NS . . . . . . . . . . . . . . . Chromosome Localization....................................................................... Conservation of Primary Sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ ....... Functional Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
527 527
GENE EXP RESSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Tran�formed Cells and Sp ecific Cell Typ es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . During Activation of Quiescent Cell to Proliferate........................................ In Termina l Dijferelltiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . In the Cell Cycle. . . . . . . . . . . . . . ............................................. .............. ......... Cis-Acting Elements in the Upstream Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PHYSIOLOGIC AL ROLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R eplicative DNA Polym erases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles in R eplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles in DNA Repair Synthesis................................................................ CONCL USIO NS AND PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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0066-4154/9110701-0513$02. 00
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PERSPECTIVES Understanding the molecular mechanisms regulating eukaryotic cell growth and division is a major endeavor in biological science. To achieve this goal it is essential to comprehend two related but separate biological problems. First, what molecular mechanisms determine whether cells should be maintained in quiescence, allowed to temlinally differentiate, or actively proliferate? Second, in actively proliferating cells, what mechanisms regulate the progres sion through stages of the cell cycle? DNA replication is essential not only for duplication of the genome but also for maintenance of genome integrity during DNA repair. Understanding the mechanisms that ensure that DNA replication does not occur in nonproliferating cells, and those that ensure that each chromosome is replicated only once during the S phase of the cell cycle, are major goals of biological science. A prerequisite to understanding the molecular mechanisms of DNA replication is a comprehension of the proteins involved in this process. Among the essential proteins for DNA replication are the DNA polymerases. Progress in studying DNA polymerases from eukaryotic cells has been problematic due to the low abundance of DNA polymerases in cells, pro teolysis during purification, and the lack of amenable genetics. Much pro gress has been made in the past few years, predominantly by the development of a cell-free reconstituted Simian virus (SV40) DNA replication system ( l , 2), and the isolation and identification o f genes and cDNAs o f DNA polymerases from Saccharomyces cerevisiae (hereafter, budding yeast) and from mammalian cells (3-8). The cell-free reconstitution of SV40 viral DNA replication provides a system for identifying polymerases that are essential for SV40 viral DNA replication and most likely function similarly in host mammalian cells. Three types of eukaryotic DNA-polymerizing enzymes have been described to date. One type of enzyme, named DNA polymerase, polymerizes DNA utilizing DNA as template; a second type, named reverse transcriptase, synthesizes DNA utilizing RNA as template; and a third type, named terminal transferase, polymerizes on the 3'OH-terminus of DNA independent of template. Five eukaryotic cellular DNA template-dependent DNA polymerases exist, designated as DNA polymerase a, {3, -y, 8, and € and encoded by distinct genes. Several recent reviews have discussed the roles of polymerases involved in cell-free replication of viral DNA (1, 2, 9, 10). Other reviews have described polymerase protein structure and en zymology in budding yeast and other higher eukaryotic cells (9, 11-13). This review focuses on the five cellular eukaryotic DNA polymerases. I discuss their properties, their associated activities and effectors, their genetic loci, their genetic structures, the conservation of their genetic and protein sequences, their functional domains, and the regulation of their
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gene expression during cell growth and through the cell cycle. The func tional role of each DNA polymerase in replication and repair synthesis is also discussed.
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NOMENCLATURE The recent identification of genes from budding yeast and mammalian cells has defined five distinct species of cellular DNA polymerases (3-8 , 14, 15). DNA polymerase a, a relatively high-molecular-weight enzyme, was the first eukaryotic DNA polymerase identified ( 1 6) . A low-molecular-weight nuclear DNA polymerase found only in vertebrates and not in budding yeast nor other lower eukaryotes is designated as DNA polymerase {3 (17, 18). The polymerase required for mitochondrial DNA replication but encoded in the nucleus is named DNA polymerase y ( 1 9-24) . This polymerase is the product of the MIl'I gene of budding yeast (15). The catalytic subunits for the three budding yeast DNA polymerases are encoded by the genes POLl, POL2, and POL3 for DNA polymerases I, II, and III, respectively. The previous Roman numeral designation of the three budding yeast DNA polymerases, Poll, PollI, and PolIII, has now been revised to polymerases a, 1:, and 6, respec tively (25). In mammalian cells, two DNA polymerases that contain tightly associated 3 '-5' exonuclease activities but respond differently to proliferating cell nuclear antigen (PCNA) for processive DNA synthesis have been de scribed (26-35). These two forms of polymerase 6 are distinct based on their dependency on PCNA for DNA synthetic processivity, and have therefore been described as PCNA-dependent and PCNA-independent polymerase 6 (33, 34). The latter was also described as DNA polymerase 6II (27, 36, 37). The PCNA-dependent polymerase 8 has now formally been designated as DNA polymerase 8, while the polymerase 811 or PCNA-independent polymerase 6 (homolog of yeast polymerase II) has now been named DNA polymeras\; E (25, 33). Much confusion was created in the literature in the past few years by naming exonuclease-associated DNA polymerases "polymerase 8." Two nuclease-associated DNA polymerase activities, one from bone marrow (38) and another 170-kDa exonuclease-associated polymerase purified from human placenta, were both described as polymerase 6 (39). However, their dependency on PCNA for DNA synthetic processivity is still unknown , and this criterion cannot be used to classify these two polymerases as either polymerase 6 or polymerase E. Another polymerase activity from calf thymus with a readily dissociable nuclease and an associ ated primase activity was described as DNA polymerase 61 ( 27, 36). It has been suggested that this represents an enzyme fraction containing polymerase a and a 3' -5' exonuclease (27, 40).
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PROPERTIES
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Protein Structures and Enzymatic Properties PROTEIN STRUCTURE The protein structure of DNA polymerase a was a controversial issue for decades. Improvement of the conventional biochemical purification procedure and development of the imunnoaffinity purification method have facilitated the resolution of the structure of this enzyme. The subunit components of DNA polymerase a isolated from a wide range of phylogenetic species either by immunoaffinity purification protocol or by conventional chromatographic methods contain a remarkably similar set of four polypeptides (12, 4 1 56) Polymerase a is composed of (a) a cluster of related high-molecular-weight polypeptides predominantly of 165- 1 80 kDa containing the catalytic function that are derivatives of a single primary polypeptide (44, 45); (b) a polypeptide of about 70 kDa with unknown catalytic function; and (c) two polypeptides of 55-60 kDa and 48-49 kDa associated with primase activity (reviewed in 57, 43, 46-48, 50, 58-70). Thus an appropriate designation for this four-subunit enzyme complex is DNA polymerase a/primase ( 1 1, 1 2) . The isolation of polymerase a full length complementary DNA (eDNA) from human cells and the gene from budding yeast defines the catalytic polypeptide as a 1 65-kDa protein (4, 5). The often observed cluster of polypeptides with a molecular mass higher than the deduced primary sequence results from posttranslational modification, while the often observed lower-molecular-mass polypeptides ranging from 1 20 to 1 65 kDa result from proteolysis of the posttranslationally modified protein at specific l abile site(s) within the N-terminus (7 1 ) . DNA polymerase f3 isolated from all vertebrate species i s a 40-kDa protein ( 1 8, 72-78). Isolation of the DNA polymerase f3 gene from rodent and human cells confirms the structure of this enzyme as a 335-amino-acid protein with a molecular size of 39 kDa ( 14, 79, 80) . A functional homolog has not yet been found in budding yeast. The protein structure of DNA polymerase l' has also been controversial due to its low abundance and proteolytic degradation during purification. Purifica tion of DNA polymerase l' from chick embryo suggested that the enzyme is a homotetramer of 47-kDa subunits (8 1 , 82) . This enzyme purified from Dro sophila melanogaster is composed of a 1 25-kDa catalytic polypeptide and an additional subunit of 35 kDa in a I : 1 ratio (23). Polymerase l' purified from Xenopus laevis contains a single polypeptide of 140 kDa reported to be associated with catalytic function (24). The confirmation that MIPI from budding yeast is the gene encoding the yeast mitochondrial DNA replication enzyme, polymerase 1', clearly defines the structure of this polymerase as a protein of 143 .5 kDa ( 15). Identification of the budding yeast POL3 (CDC2) gene encoding DNA -
.
EUKARYOTIC DNA POLYMERASES
5 17
polymerasf� () defines the molecular mass of the catalytic subunit of this polymerasf� as 125 kDa (6). B iochemical purification of polymerase () activity
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from calf thymus and HeLa cells demonstrates an enzyme composed of a 125-kDa catalytic polypeptide and an associated 48-kDa subunit of unknown function (29, 33). Polymerase E purified from HeLa cells was reported to be a protein of 2 15
kDa (32, 33) . DNA polymerase E (polymerase II) from budding yeast was described as a group of proteins ranging in molecular size from 132 to 200 kDa containing polymerase activity (83, 84). The recently isolated gene for polymerast: E from budding yeast encodes a protein of 255 kDa that is by far the largest DNA polymerase yet described (8). It is assumed that the pre viously observed lower-molecular-mass proteins from either mammalian cells or yeast were proteolytically degraded products of the 255-kDa polypeptide. Two polypeptides of 80 and 34 kDa from yeast polymerase E enzyme prepara
tions have been provisionally assigned as subunit components of this enzyme (A. Sugino, personal communication). In contrast, polymerase E purified from HeLa cells does not contain these subunits (32, 33) . The general enzymatic properties such as primer template preference, divalent cation preferences, salt sensitivity, and in hibitors of these five distinct polymerases have been described by others (13, 18, 29, 83, 85). Properties that can be used to distinguish these DNA ENZYMATIC PROPERTIES
polymerases are summarized in Table 1 and are not discussed here in detail. Two notable enzymatic properties of DNA polymerases are processivity and fidelity. Processivity of a polymerase is the extent of DNA chain el onga tion by DNA polymerase per interaction with the 3' OH-end of the DNA
primer. Processivity of DNA polymerases a and f3 has been studied ex tensively. For DNA polymerase a, depending on the assay conditions , purity of the enzyme, integrity of the catalytic polypeptide, and the subunit com ponents of the enzyme fraction used, a large variation in the number of nucleotides. polymerized per binding event has been reported (86-88). Com pared to the other four eukaryotic DNA polymerases, polymerase a is gener ally regarded as moderately processive (Table l ) . Despite the dependency of
processivity on reaction conditions and polymerase a integrity, processivity has been used as a parameter to identify a variety of effectors and accessory proteins for polymerase a, which have been reported to enhance its processiv ity (89 -92). With the exception of an accessory protein RF-A, the in vivo significance of these identified effectors is not yet clear. DNA polymerase f3 is the smallest DNA polymera se in eukaryot ic cel ls,
but it has several unique and distinct enzymatic properties. It is insensitive to aphidicolin, sulfhydryl reagents, salt, and butylphenyl-dGTP ( 18, 93), but is sensitive to ddNTPs (18, 86). Processivity of polymerase f3 depends on the
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Table 1
Eukaryotic DNA polymerases: protein structure, cellular location, and properties DNA polymerase
{3
'Y
8
165"
40
140
125
255
70 58 48
none
unknown
48
unknown
Protein structure catalytic polypeptide (kDa) associated subunits
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(kDa)
Yeast gene
POLl none (CDC] 7)
MIPl
POLJ (CDC2)
POL2
Cellular location
nuclear
nuclear
mito
nuclear
nuclear
Associated enzyme activities
3' -5' exonuclease
noneb
none
yes
yes
yes
primase
yes
none
none
none
none
med
low
high
low
high
Properties processivity inherent
high
with PCNA fidelity
high
low
high
high
high
yes
Inhibitors aphidicolin
yes
no
no
yes
N-ethylmaleimide
yes
no
yes
yes
unknown
butylphenyl-dGTP
yes
no
no
no
no
dideoxynuc1eoside 5' -triphosphate
no
yes
med
no
no
no
unknown
unknown
yes
med
(ddNTP) carbonyldiphosphonate
Molecular mass deduced from primary protein sequence. b Cryptic exonuclease in the isolated catalytic polypeptide was reported in Drosophila melanogaster embryo
a
(102).
metal ion in the reaction; it is five times higher in reactions with Mn2+ than in reactions with Mg2+ as activator
(94).
Polymerase 'Y is aphidicolin resistant but N-ethylmaleimide sensitive and is moderately inhibited by dideoxythymidine triphosphate (ddTTP) merase 'Y has been reported to be highly processive Polymerases
{) and
E
(24). Poly in DNA synthesis (18).
are distinguished from the other DNA polymerases by
their readily detectable proofreading 3' -5' exonuclease activity. Both are aphidicolin sensitive
(29, 32, 33, 36), and both are rather insensitive to the
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inhibition of butylphenyl-dGTP. Furthermore, while polymerase 8 is stimu lated by 10% dimethyl sulfoxide (DMSO), polymerase € is inhibited by this reagent (33). These two DNA polymerases are immunologically and structur ally distinct from polymerase a, but have some immunological and structural similarity with each other (34) . Interestingly, these two polymerases are strikingly distinct in their processivity. Polymerase 8 has low processivity with long single-stranded templates, but, in the presence of peNA, polymerase 0 can perform highly processive DNA synthesis on this template. Thus, PCNA has been designated as the auxiliary protein of DNA polymerase 8 (95). In contrast to polymerase 8, DNA polymerase E has an inherently high processivity on this template in the absence of peNA in reactions with low concentrations of Mg 2+ (30, 32, 3 3 , 84) . DNA synthetic fidelity and mutation specificity of polymerases a, {3, and 'Y have been studied extensively (reviewed in 1 8 , 96-99). A detailed discussion of fidelity studies and the mutation specificity of each polymerase is beyond the scope of this review. A summary of these studies is presented in Table 1. Several interesting findings are noteworthy. Despite the lack of a measurable proofreading 3 ' -5 ' exonuclease in polymerase a/primase complex , the mis incorporation rate of this enzyme is low. Two factors are suggested to influence the fidelity of polymerase a. First, the more intact form of the four-subunit complex of DNA polymerase l1'/primase has higher DNA syn thetic accuracy than aged and proteolyzed enzyme ( 1 00, 1 0 1 ). A second factor is the presence of a proofreading exonuclease activity. A cryptic 3' -5' exonuclease activity in the large catalytic subunit of polymerase a from D . melanogaster embryo has been described when this subunit i s separated from the 70-kDa subunit ( 1 02). This unmasked cryptic exonuclease in the catalytic polypeptide enhances the fidelity of DNA synthesis 1 00-fold. Furthermore, this separated polymerase a catalytic polypeptide with the cryptic exonucle ase has altered sensitivity to inhibitors. Like polymerases 8 and E, it hecomes less sensitive to butylphenyl-dGTP , whereas this analog is a potent inhibitor of the intact polymerase a/primase complex ( 1 03) . Moreover, a polymerase a enzyme preparation from human lymphocyte containing a loosely associated proofreading exonuclease was reported to have higher DNA synthetic fidelity ( 1 04) . Consistent with the notion that proofreading exonuclease enhances polymerase DNA synthetic fidelity, the fidelity of calf thymus DNA polymerase E (previously described as polymerase 811) has an accuracy 1 0and 500-fold greater than that of polymerases a and {3, respectively (37).
Catalytic Mechanisms Using steady-state kinetics analysis and supplemented by direct physical measurement, the catalytic mechanisms of DNA polymerases a and {3 were
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thoroughly investigated (reviewed in 105 and 45,86,94, 105-Ill). Exten sive studies of DNA polymerase a and substrate interaction were performed
with the biochemically purified but proteolyzed form of the enzyme ( 1 1 2) . Most o f these polymerase a and substrate interaction mechanisms were later
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proven to be qualitatively identical when reassessed with the immunopurified polymerase a/primase complex (44) . In a separate study, analysis with im munoaffinity-purified calf thymus DNA polymerase a demonstrated quali tatively higher affinity for deoxynucleoside 5' -triphosphates (dNTPs) and
primer-template (52). These studies suggest that the intact form of DNA polymerase
a
might
yield quantitatively more efficient enzyme for the assessment of catalytic mechanism. The interaction of polymerase a and substrates was found to obey a rigidly ordered sequential ter mechanism. The order of the sequential ler mechanism is first, interaction with template (single-stranded DNA),
second, interaction with primer stem, and third, interaction with dNTP. Specification of the addition of dNTP to the enzyme is determined by the template sequence. A minimum length of 8 nucleotides of primer is required for effective binding with polymerase a. The terminal 3-5 nucleotides of the primer must be template-complementary . A single mispaired terminal nucleo tide prevents correct primer binding and the subsequent step of dNTP addition to the enzyme ( 106- 1 1 0). This finding was later confirmed by assessment of rate of extension from mispaired primers by calf thymus polymerase a ( 1 1 3 , 1 14). The rate o f extension from mispairs is 103 to 106 times slower than from a paired primer terminus ( 1 14). Hydrolysis of the mispaired primer residue by either the f subunit of Escherichia coli polymerase III or the proofreading 3' exonuclease associated in polymerase {) permits subsequent extension and increases the fidelity of DNA polymerase a ( 1 1 5 , Il6). 3'-Terminal H or OH, but not P04, can interact with polymerase a. The primer binding occurs through the coordinated participation of four Mg 2+ -primer binding subsites, which may serve as a shuttle to facilitate not only phosphodiester bond formation but also polymerase translocation. The catalytically active
polymerase
a
appears to have two positively cooperative single-stranded
DNA-binding sites. Thus, it was proposed that polymerase a is a con formationally active enzyme that responds to signals from template sequence that are transduced via template-binding site(s) interaction ( 1 09, 1 10). Unlike polymerase a, DNA polymerase {3 has a weak affinity for single stranded DNA that is not modulated by base sequence and not enhanced by the presence of potentially base-paired 3' -OH termini. Polymerase f3 has no affinity for intact duplex DNA, but has a high affin ity for nicked duplex DNA with nicked termini either bearing 3' -OH or 3' -P04 residues. Polymerase f3 can perform a limited strand displacement synthesis at the 3 ' -OH termini of the nicked DNA ( 1 1 1). In corroboration of these earlier findings , polymerase
EUKARYOTIC DNA POLYMERASES
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{3 from HeLa cells was also demonstrated to perform limited strand displace ment synthesis on 3' -OH termini in DNA produced by AP endonuclease at the 5' side of apurinic sites. Polymerase {3 is able to insert a single com plementary nucleotide into the single nucleotide gap created (117).
Polymerase {3 purified from human cells has less stringent requirements for primer interaction than polymerase a. Polymerase {3 is reactive with primers that contain 1-3 mispaired terminal residues. Polymerase {3 purified from mouse cells was reported to carry out a two substrate-two product reaction that follows ordered bi-bi kinetics: interacting with primer template first, followed by dNTP ( 1 1 8) . The mechanism of substrate interaction of human polymerast! f3 is also rigidly ordered, and sequential by binding to DNA first followed by dNTP. However, binding to primer-template appears to occur by a concerted mechanism. The primary signal for catalytic productive interac tion of polymerase {3 with DNA is a base-paired primer moiety that must be adjacent to a short length of potentially single-stranded template. The mini mum length of this template and processivity are influenced by the divalent cation. With Mg2+ , minimum template length is >5 nuc1eotides and polymerization is distributive, whereas, with Mn2+ , the minimum required template is a single nucleotide and the polymerization is more processive by inserting 4-6 residues per cycle of primer interaction (94, Il l) . Consistent with the notion that higher processivity is responsible for higher fidelity, the error rate at hot spots for polymerase {3 was also found to be lower in reactions with Mn2+ as cation ( 1 19) . ASSOCIATED ACTIVITIES AND EFFECTORS
Primase All DNA polymerases require a 3' -hydroxyl terminus of a preexisting primer for reaction ( 120, 121). DNA polymerase a is the only eukaryotic polymerase with a tightly associated primase. Primase activity resides in a heterodimer of 55-60 kDa and 48--49 kDa polypeptides that have been purified from various biological systems as the associated activity and subunit component of polymerase a. The purification, enzymatic properties, and reaction mech anisms of primase reported from numerous laboratories have been reviewed (57). Some: recent developments that were not included in the previous review are discussed here. The genes for both primase subunits from budding yeast are now cloned and designated as PRJ1 and PR12. By gene disruption, both genes were found to be essential (69, 122, 123). By affinity labeling, the ATP- or GTP-binding site(s) of primase from calf thymus and yeast were found to be localized exclusively in the 48-kDa subunit (67 , 124). A monoclonal antibody against
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the 60-kDa subunit of chick primase has been produced. Immunofluorescence demonstrates the exclusive nuclear localization of primase in granular struc tures bound to the nuclear matrix. Furthermore, primase antigen was reduced in quiescent cells and induced when quiescent cells were activated to pro liferate by serum addition. These results suggest that the expression and organization of primase in the granular structures on the nuclear matrix are regulated in correlation with cell proliferation (125). The cDNA of the 49-kDa subunit of mouse primase was cloned and characterized (126). Se quence comparison with yeast primase 49-kDa subunit shows a high con servation of amino acid sequences in the N-terminal halves of the polypeptides and a potential metal-binding domain (Zn finger) in this region. The C-terminal region diverges from yeast primase. During serum induction of quiescent cells to proliferate, expression of the mouse 49-kDa primase gene lO-fold at the steady state transcript level, but in is positively induced actively proliferating cells the gene is constitutively expressed throughout the cell cycle (127). This finding is in agreement with the immunofluorescence data reported for chick primase (125). The cDNA for the 59-kDa large subunit of mouse primase has also been cloned (B. Y. Tseng, personal communica tion). �
3' -5' Exonuclease None of the five cellular DNA polymerases has an associated endonuclease or 5'-3' exonuclease. Polymerases y, S, and € do have an associated proofread ing 3'-5' exonuclease. The proofreading 3'-5' exonuclease and polymerase y activity have been suggested to reside in the same polypeptide. From X. iaevis, the 3' -5' exonuclease activity and polymerase y activity copurified in a constant ratio in the final three steps of purification. The two enzymatic activities also cosediment in a glycerol gradient under partially denaturing conditions (128). In chick embryo, the 3' -5' exonuclease was found to coprecipitate with DNA polymerase y during immunoprecipitation with anti DNA polymerase yantibody (129). Like the classic proofreading nuclease of E. coli polymerase I and T4 polymerase (130, 131), the 3'-5' exonuclease of polymerase y hydrolyzes mismatched termini with significantly greater effi ciency than base-paired termini (128, 129). The 3' -5' exonuclease activities in polymerases {) and E also behave as typical proofreading nucleases with the preference for hydrolyzing mismatched over base-paired termini (32, 83, 84, 132). As with the proofreading 3' -5' exonuclease in prokaryotes, budding yeast polymerase 8 also has a demonstrable turnover of dTTP to dTMP by the successive actions of polymerase and exonuclease (132). Based on the in hibitor studies and the constant ratio of nuclease to polymerase activity in the final steps of purification, it is suggested that the 3' -5' exonuclease activity of both polymerases S and € reside in the same polypeptide as the polymerase
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EUKARYOTIC DNA POLYMERASES
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activity (26, 38 , 83, 84) . As described above, a cryptic 3' -5' exonuclease was also reported in the catalytic polypeptide of Drosophila polymerase a when isolated from the other subunits ( 1 02). An 3' -5' exonuclease activity has been described jin recombinant yeast polymerase a 1 80-kDa subunit recently ( 1 32a) . This 3'-5' exonuclease activity is not a conventional proofreading nuclease, however. The yeast 1 80-kDa subunit, as well as the 1 80-kDa/86kDa complex and the four-subunit polymerase a/primase complex, was cap able of removing 3' terminal nucleotide of poly(dT)6oo- 32p-dNMPo.4-0.5' The physiological significance of this yeast 1 80-kDa associated exonuclease is not yet known. The catalytic polypeptide of human DNA polymerase a has been overproduced from recombinant baculovirus-infected insect cells as a single polypeptide of 1 80 kDa. This single-polypeptide polymerase a does not have a detectable proofreading 3' -5' exonuclease activity when assayed with either mismatched or matched base-paired termini (W. Copeland and T. Wang, manuscript submitted). It is interesting that the cryptic proofreading exonucle ase of DNA polymerase a appears to be unique for the embryo of Drosophila.
Proliferating Cell Nuclear Antigen (PCNA) An auxiliary protein for DNA polymerase 8 that enhances its processivity was found to be immunologically , structurally , and functionally identical with proliferating cell nuclear antigen, PCNA (29, 95, 133, 133a, 1 34). PCNA is a 36-kDa nuclear protein reactive with sera from a subset of patients with the autoimmune disease systemic lupus erythematosus (SLE), and this protein was only found in actively proliferating cells ( 1 35 , 1 36) . PCNA was also found to be essential for in vitro SV40 DNA replication (133, 1 37). Reviews of the role of PCNA in viral and cellular DNA replication and cell growth have been published recently (2, 1 3, 1 38). The gene and cDNA for PCNA have been isolated and characterized from rat , human, budding yeast, and Drosophila. Protein sequence comparison indicates that PCNA is a highly conserved protein among these eukaryotes ( 1 39-1 43) . Budding yeast PCNA gene (POL30), like the budding yeast polymerase 8, is periodically expressed at the level of steady-state mRNA in starve-feed cultures . During meiosis, the mRNA level also increases prior to initiation of premeiotic DNA synthesis ( 14 1 ) . In contrast to budding yeast, the protein level of PCNA in mammalian cells was reported to be constitutively expressed throughout the cell cycle, although PCNA mRNA and protein levels were dramatically increased during activation of quiescent cells to proliferate ( 1 40, 1 44). PCNA does not appear to have a stringent species specificity for i ts interaction with polymerase 8 and other replication factors . Calf PCNA can stimulate yeast polymerase 8 ( 1 45); calf thymus PCNA and human RF-C (a replication factor, see discussion below) are able to stimulate budding yeast
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polymerase S DNA synthesis on singly primed M 1 3 single-stranded DNA in an ATP-dependent manner ( 1 46). Drosophila PCNA could substitute for human PCNA, although with reduced efficiency, in stimulating SV40 in vitro DNA replication ( 143). PCNA has also been shown to affect the DNA synthetic activity of polymerase E. Under certain salt conditions in the pres ence of RF-C, calf thymus PCNA can enhance budding yeast polymerase E DNA synthetic activity on singly primed single-stranded DNA and can en hance its synthetic processivity in homopolymer reactions (84, 1 46) . In SV40 origin-dependent DNA replication, the absence of PCNA yields short and lagging-strand-specific products, which are predominantly derived from re gions around the SV40 origin ( 1 37). PCNA also stimulates the ATPase activity of RF-C and is an essential component of the primer recognition complex, PCNA/RF-ClATP (92) . Evidence from product analysis of the SV40 in vitro replication reactions suggests that PCNA is the functional equivalent of T4 phage gene 45 protein or E. coli DNA polymerase III f3 subunit ( 1 47). The functional analogy between PCNA and T4 phage gene 45 protein is also reflected in the primary amino acid sequences of these proteins. These two proteins have a 3 1-50% primary sequence similarity within a limited region, but have no significant similarity to dnaN gene product, which encodes the f3 subunit of E. coli DNA polymerase III holoenzyme ( 1 47) . In sum, several lines o f evidence support the conclusion that PCNA does not directly affect the DNA synthetic activity of DNA polymerase a. For polymerase S, synergistic interaction of PCNA with RF-C/ATP and RF-A is required for the optimal DNA synthetic activity on primed single-stranded DNA or in reconstituted SV40 origin containing DNA reaction. Depending on the reaction condition and the presence of RF-C and ATP, the conjecture is that PCNA might also have an effect on in vitro DNA synthetic activity of polymerase E (84, 146).
Replication Factor A (RF-A) RF-A (also named replication protein A, RP-A, and human single-stranded DNA-binding protein, HSSB) is a multisubunit single-stranded DNA-binding protein. RF-A contains three polypeptides of 70, 34, and 1 1 kDa and func tions as an auxiliary protein for both polymerases a and S. RF-A is required for the initiation and elongation stages of in vitro SV40 DNA replication ( 148- 153). Biochemical and immunological evidence indicates that the sing le-stranded DNA-binding activity resides exclusively in the 70-kDa subunit ( 149, 1 5 1 , 1 54) . RF-A alone stimulates DNA polymerase a activity on either primed M 1 3 DNA or oligo dT-primed polydA , resulting in short DNA products; PCNA had no effect on the size of the product. In contrast to the effect on polymerase a, RF-A alone has no specific effect on the reactivity of
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EUKARYOTIC DNA POLYMERASES
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polymerase;; even in the presence of PCNA . However, polymerase;; can be synergistically stimulated by PCNA, RF-A, and RF-C (another replication factor, see discussion below), yielding long DNA products (152-154) . Immunological evidence has suggested that the 70-kDa subunit may be required for DNA unwinding and polymerase S stimulation, whereas both 70and 32-34-kDa subunits have been implicated to be involved in the stimula tion of polymerase a DNA synthetic activity (154). The gene for the 70-kDa subunit has been cloned from budding yeast and found to be essential (155). The deduced protein sequence reveals significant amino acid similarity of this yeast 70-kDa RF-A subunit with its human homolog (155). The cDNA for the 34-kDa subunit of RF-A from human cells was also cloned and overexpressed (156). The 34-kDa protein is a phosphoprotein, and phosphorylation of this subunit is cell cycle regulated with higher phosphorylation occurring at late 01 and early S phase (157) . Yeast RF-A or other single-stranded DNA binding proteins, including E. coli single-stranded binding protein (SS B) and adenovirus and herpes simplex virus DNA-binding proteins, are able to replace human RF-A in the unwinding step in the initial stage of SV40 in vitro DNA replication. However, yeast RF-A and these other SSBs are unable to substitute for human RF-A in the SV40 in vitro DNA replication reaction (1, 2, 153, 158). Thus, like polymerase a, RF-A demonstrates stringent species specificity iln SV40 in vitro DNA replication . It also appears to be involved in genetic recombination (155).
Replication Factor C (RF-C) RF-C (also named activator I, AI) is a multi subunit protein complex that has primer/template binding and DNA-dependent ATPase activity . The intrinsic DNA-dependent ATPase activity of RF-C is stimulated by PCNA (147), and addition of RF-A further stimulates the DNA-dependent ATPase activity (159) . Purified RF-C is composed of subunits of 140, 41, and 37 kDa. The 140-kDa subunit binds primer/template DNA, whereas the 41-kDa subunit binds ATP (92). In SV40 in vitro DNA replication reactions, the absence of RF-C causes accumulation of early replicative intermediates and yields DNA products that hybridize only to the lagging-strand template (92, 152, 160163). This observation suggests that RF-C has a profound effect on leading strand DNA synthesis. RF-C appears to be the functional equivalent of the T4 phage gene 44/62 complex (reviewed in 164 and 165). Similar to the T4 phage DNA replication system, processive DNA synthesis by polymerase 8 on primed M13 single-stranded DNA requires RF-A, RF-C, and PCNA and hydrolysis of ATP or dATP (92, 147, 159, 162, 163, 166) . Furthermore, replication complexes from (a) T4 phage containing gene products 43, 44/62 and 45, or T7 polymerase/thioredoxin complex, (b) E. coli DNA polymerase III holoenzyme, and (c) other DNA polymerases can substitute for the lead-
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ing-strand polymerase complex from human cells in SV40 in vitro DNA replication reaction ( 1 62, 1 68). Extensive product analysis of reactions with RF-C in various combinations with PCNA, ATP, and RF-A, in conjunction with isolation of the replication protein complex by gel filtration, and analysis of the kinetics of protein complex formation, support a model of protein-protein interaction hierarchy at the replication fork. A model has been proposed that binding of RF-C to the lagging-strand polymerase complex at the replication origin occurs first, followed by binding of PCNA in an ATP-dependent manner for the formation of a "primer recognition complex," which in cooperation with RF-A plays a crucial role in initiating the leading-strand synthesis from the nascent DNA fragment synthesized by the lagging-strand polymerase complex (92, 1 62, 1 63 , 1 66). A proposed model showing the effect of RF-A, RF-C, and PCNA on the lagging-strand and leading-strand switching is illustrated in Figure 3 and discussed further in the section of this review on Physiological Roles.
T Antigen In SV40 viral DNA replication, it is reasonable to assume that there are protein-protein interactions between T antigen and cellular replication pro teins during initiation of the SV 40 DNA replication. An earlier report de scribed the association of DNA polymerase a activity from crude cell extracts with T antigen, but purified polymerase a/primase complex failed to demon strate significant interaction with T antigen ( 1 69). These observations suggest that the interaction between T antigen and polymerase a/primase complex might be due to a third yet unidentified cellular protein in the crude extract. A subset of T antigen monoclonal antibodies was shown to prevent the binding of T antigen and polymerase a/primase from crude cell lysate, and in terestingly these antibodies also block T antigen interaction with murine p53 protein (170, 171) . Recently, this issue has been investigated further with the highly purified four-subunit polymerase a/primase complex and T antigen. It has been demonstrated that T antigen binds directly and specifically to the catalytic subunit of human DNA polymerase a. The amino-terminal 83 amino acids of T antigen are both necessary and sufficient for the binding. Further more , polymerase a from human tissue has a lO-fold higher affinity for T antigen than does polymerase a from calf, and conversely human polymerase a that binds the SV40 T antigen with high affinity does not bind polyoma T antigen ( 1 72; I . Domeiter, E. Fanning , personal communication) . This is consistent with an earlier report of species specificity of polymerase a/ primase in SV40 in vitro DNA replication reaction ( 1 73). Thus, T antigen can be classified as another effector for DNA polymerase a in the viral SV40 replication system, perhaps by directing the polymerase to the origin of replication, where T antigen is bound.
EUKARYOTIC DNA POLYMERASES
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Several other effectors for polymerase 0' have been reported. Among those, two proteins named Cl and C2 can increase the affinity of DNA polymerase 0' to primer stem 20-30-fold. Interestingly, the effect of these two proteins on polymerase 0' also demonstrates species specificity ( 174, 1 75). Another protein, alpha accessory factor (AAF) , is highly specific for polymerase 0' with no effect on polymerases f3, 8, or y. AAF increases the affinity of polymerase a/primase for DNA templates, enhances its processivity, and aIlows this enzyme to traverse double-stranded DNA region ( 1 76, 177). GENETIC STRUCTURE AND PREDICTED FUNCTIONAL DOMAINS
Chromosome Localization The catalytic polypeptide of human DNA polymerase a has been mapped to the short arm of the X-chromosome at Xp2 1 .3 to Xp22. 1 by expression, and later confirmed by genomic Southern hybridization with the cDNA (4, 1 78). The gene for mouse polymerase a catalytic polypeptide was recently localized on the mouse X-chromosome in region C-D ( 1 79). The genes of the two mouse primase p49 and pS8 subunits were localized to mouse chromosomes 10 and 1 , respectively ( 1 79) . The genes coding for all four subunits of budding yeast DNA polymerase 0' were mapped. The catalytic polypeptide maps on yeast chromosome XIV, the 74-86 kDa subunit on chromosome II , and the two primase 58- and 48-kDa subunits on chromosomes XI and IX, respectively ( 1 80) . The chromosome localization of budding yeast polymerase 8 is mapped to the left arm of chromosome IV (6) , whereas the chromosome localization of polymerase E is not yet known. The yeast MIP1 gene encoding mitochondrial DNA polymerase y was mapped to the right arm of yeast chromosome XV ( 1 5). The genes for human and mouse DNA polymerase f3 were both mapped to chromosome 8 of human and mouse, respectively ( 1 8 1- 1 83). Further fine mapping of human and mouse DNA polymerase f3 gene has localized the gene near localization of the tissue plasminogen activator on the proximal short arm of chromosome 8 ( 1 84) .
Conservation of Primary Sequences Sequence comparison of cDNAs and genes of various prokaryotic and eu karyotic DNA polymerases has revealed an evolutionary conservation of three groups of DNA polymerases on the basis of their amino acid sequence relatedness: (a) E. coli DNA polymerase I and phage T7 DNA polymerase ( 1 85); (b) DNA polymerase f3 and terminal transferase (80, 1 86); and (c) a-like DNA pulymerases, including protein-primed replicative DNA polymerases (4, 1 87).
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WANG
Comparison of deduced amino acid sequences of DNA polymerase {3 and other proteins showed a sequence similarity with human terminal de oxynucleotidyltransferase (80, 1 86). Sequence alignments revealed conserva tion between residues 42-215 of polymerase f3 and residues 195-366 of terminal transferase. Nearly 30% of these 1 74 amino acid residues of polymerase f3 were identical with those of the 1 72-residue sequence of terminal transferase. Forty residues (23%) represented conservative substitu tion. Thus, the sum of identical residues, the favored substitutions , and the conservative substitutions in amino acid pairs was 1 24 out of 1 74 residues (7 1 . 3%). The overall predicted secondary structures of the two enzymes were also closely similar in their homologous regions (80) . However, the C terminal region (amino acid residues 2 1 6-335) of DNA polymerase f3 has no significant similarity with that of terminal transferase. Interestingly, the similar region is coded exclusively by the third and the later exons. The isolation of the catalytic polypeptide of human DNA polymerase a cDNA and analysis of its sequence revealed striking sequence similarity with replicative DNA polymerases from both prokaryotes and eukaryotes (4, 1 87). Furthermore, by utilizing both immunologic and molecular genetic approaches, the differential evolutionary conservation of four unique epitopes on the catalytic polypeptide and the conservation of genomic DNA sequences have been demonstrated among organisms as phylogenetically disparate as primate and unicellular fungi ( 1 88). These findings have facilitated the identification of many DNA polymerase genes and cDNAs and have led to the recognition of an a-like family of DNA polymerase genes. From amino acid residues 609 to 1085 of human DNA polymerase a , six regions exist that are similar to regions of the three budding yeast DNA polymerases a, 15, and E, a potential budding yeast polymerase gene, REV3, product that is involved in mutagenesis responses, DNA polymerases from the herpes simplex virus (HSY) family, cytomegalovirus (Hey), Epstein-Barr virus (EBV), and vac cinia virus polymerase (4-6, 8 , 45 , 1 87 , 1 89-1 93). Five regions of similarity exist to adenovirus DNA polymerase and bacteriophage T4 DNA polymerase ( 1 94, 1 95). Three regions of similarity exist to DNA polymerases from Bacillus subtilis phage 29 and E. coli phage PRDI ( 1 96, 1 97), as well as to two potential DNA polymerases, yeast linear plasmid pGKLI and maize SI mitochondrial DNA ( 198-200) . The regions described above are designated I to VI according to their extent of similarity, with region I being the most conserved and region VI, the least similar. The significance of these six regions is further underscored by their relative linear spatial arrangements within each polymerase polypeptide (Fig ure 1). The order of these six regions in each polymerase polypeptide is IV -11 -VI-III-I-V, while the distances between each of these conserved regions are variable. While regions I, II, and III appear to be the basic core regions of this a-like DNA polymerase family, yeast MIPI gene encoding the yeast
Annu. Rev. Biochem. 1991.60:513-552. Downloaded from www.annualreviews.org Access provided by Mahidol University on 01/20/15. For personal use only.
EUKARYOTIC DNA POLYMERASES
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polymerase 'Y revealed the presence of regions I, II, and VI but not regions III, IV, and V. Furthermore, the linear spatial order of the three regions, I, II, and VI, is different from that in the other a-like family DNA polymerases (15). Although the sequence of yeast polymerase € demonstrates the presence of these six conserved regions, and the linear spatial arrangements of these regions are similar to those of the other a-like DNA polymerases, the similarity among these six regions is relatively weak. Region I, containing sequence -YGTDTS-, which is the most highly conserved sequence among the a-like family DNA polymerases, is poorly conserved in yeast polymerase € (8). Both yeast DNA polymerases 8 and € also showed distinct sequence similarity to viral polymerases (6, 8). This suggests that these two 3' -5' exonuclease-containing DNA polymerases of yeast might belong to a sub family of a-like polymerases. Recently the dinA gene of E. coli was found to encode the E. coli DNA polymerase II and, interestingly, the sequence of this gene revealls the presence of regions IV, II, III, I, and V sequences of the a-like family polymerase (20 1 ) . The presence of these conserved regions in DNA polyrnerases from human to bacteriophage suggests that this group of DNA polymerases may all have been derived from a common primordial gene. Human DNA pol a
IV
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Vaccinia Virus DNA pol
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pGKl1 S1 Mitochondria
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