ANNUAL REVIEWS
Further
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1991. 60:477-511
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FIDELITY MECHANISMS Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
IN Dl'IA REPLICATION Harrison Echols Department of Molecular and Cell Biology, University of California, Berkeley, California
94720
Myron F. Goodman Department of Biological Science, University of Southern California, Los Angeles,
California 90089-1340 KEY WORDS:
DNA polymerases, mutation mechanisms, DNA damage, editing
by DNA
polymerases.
CONTENTS PERSPECTIVES AND SUMMARY ............................................................. .
478
BASE INSERTION SELECTIVITY BY DNA POLYMERASES ......................... .
479 479
The Base 1''lSertion Pathway ................................................................... . Structural Aspects to Base Selection by DNA Polymerases ............................. . Polymerase Insertion Errors ................................................................... . Fidelity Assays and Major Conclusions ..................................................... . Error Avoidance in Base Insertion----General Conclusions ..... ........................ . EDITING BY DNA POLYMERASES........................................................... .
Exonucleolytic Editing by Prokaryotic Polymerases ...................................... . Editing in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetic Amplification of Editing: Inefficient Extension from a Mismatched 3'-Terminus.......................................................... . Error Avoidance in Editing----General Conclusions ....................................... . Possible Additional Editing Mechanisms: the MutT Pathway ........................... . MUTATIONS GENERATED BY DAMAGE TO DNA ..................................... .
Response of DNA Polymerase to DNA Lesions ............................................ . The SOS R"sponse of Escherichia coli ....................................................... Multiple Roles of RecA in the SOS Response .............................................. . Some Biological Comments .................................................................... .
480 482 483 488 491 491 492 493 494 496 497 497 499 500 505
477 0066-4 1 54/91/0701 -0477$02.00
478
ECHOLS & GOODMAN
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
PERSPECTIVES AND SUMMARY The survival of an organism requires precise duplication of the genome. Error frequencies are typically only 10-9 to 10 10 per base replicated. The bio chemical problem posed by this demand for faithful duplication is ex ceptionally complex because the correct (Watson-Crick) base pairs do not exhibit large structural or energetic differences from some incorrect base pairs (e. g . G·T vs A·T) . For the well-defined prokaryotic replication systems , the requirement for a low mutation rate is achieved by the sequential operation of three fidelity mechanisms: (a) selection of the correct deoxynucleoside triphosphate (dNTP) substrate in the polymerization reaction (base selection); (b) exonucleolytic removal of an incorrectly inserted deoxynucleoside monophosphate (dNMP) from the end of the growing chain (editing); (c) postreplicative excision of an incorrectly inserted deoxynucleoside monophosphate after the polymerase has extended the DNA chain (mismatch repair). Each of these mechanisms exhibits an impressive selectivity for excluding noncanonical base pairs; the sequential application of all three fidelity operations yields precisely duplicated DNA. For eukaryotic replica tion systems, only base selection has so far been clearly defined as a general fidelity mechanism. However, most cellular and viral eukaryotic polymerases exhibit exonucleolytic editing activities , and there has been excellent recent evidence for mismatch correction pathways. Thus the prokaryotic solutions described here are likely to be general . In this review we consider in detail only the two coupled fidelity mech anisms that involve the direct participation of the DNA polymerase-base selection and editing. Excellent recent reviews have appeared on mismatch repair (1-3). We focus on the fundamental biochemical question: how does a DNA polymerase distinguish a correct from an incorrect base pair with such astounding precision? Recent structural and kinetic data indicate that polymerases achieve selectivity by mechanisms that differ in detail . However, from the available evidence, we believe that all polymerases follow general fidelity principles for base selection and editing. For base selection, the most likely discrimination principle is an exquisite demand for the precise geometry of the Watson-Crick base pair. The most remarkable structural property of the A'T and G'C base pairs is their complete geometric equivalence as subunits of the double helix; all other non-Watson Crick base pairs known to occur (e. g. the G·T "wobble" pair) have altered geometry . We presume that the active site of the polymerase exerts transition state selectivity for the identical geometry (CI' distances and bond angles) of the Watson-Crick base pairs. As a consequence, the correct dNTP is used 104 or 105 times as efficiently as an incorrect dNTP. This demand for Watson Crick geometry at the transition state might be manifested either in more rapid dissociation of incorrect dNTP, or slower phosphodiester bond formation for
FIDELITY MECHANISMS IN DNA REPLICATION
479
misoriented bases , or most likely both. The active site of a polymerase probably evolved to catalyze phosphodiester bond formation with either A, T ,
G, o r C i n the dNTP site, with frequency dependent o n pairing energetics between substrate and template. Thus a geometric selection mechanism could be achieved by fine-tuning the allowed template-substrate configurations . For exonucleolytic editing , the most likely discrimination mechanism is the "melting capacity" of the mispaired DNA; a misaligned base at the 3' end will be more often in a single-strand configuration. This mechanism is an easy
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
evolutionary transition from a free single-strand exonuclease. However, the degree of discrimination against a mispair is likely to be relatively small, especially with respect to the more thermally sensitive A'T correct pairs. Editing bec:omes an efficient process, with discrimination of 102 or more , because of kinetic amplification mechanisms. In the most clearly defined mechanism, the next base addition reaction following a misinsertion is slow compared to base addition following a correct insertion. Thus the exonuclease has longer to act on the mispair, amplifying the editing discrimination. Certain DNA lesions inhibit DNA replication. The damage site is presum ably suffici(!ntly distorted that the geometric recognition mechanism does not allow effective base insertion. In addition, the editing exonuclease will preferentiaHy remove even a "correctly" inserted base because the resultant distorted base pair looks like a mismatch. The rescue of such a stalled polymerase requires a complex cellular response. In the last section of this review, we consider briefly the induced "SOS response" to replication blocking lesions. The SOS response provides for a highly mutagenic pathway of "translesion" DNA replication, dependent on induced synthesis of the mutation proteins UmuC and UmuD, followed by regulated processing of UmuD to its active form, UmuD'. An interesting point is that mutations are introduced by an induced and highly regulated pathway; SOS mutagenesis is not a passive response to DNA damage. The existence of an induced mutation pathway might seem puzzling in view of our opening statement that the mutation rate must be kept low. However, mutation is the source of genetic variation, and a regulated loss of replication fidelity might serve to accelerate evolutionary adaptation in an endangered population. Thus the fidelity of DNA replication may be subject to environmental regulation.
BASE INSERTION SELECTIVITY BY DNA POLYMERASES
The Base Insertion Pathway The role of a D NA polymerase is to catalyze the formation of phosphodiester bonds between primer 3' -temlini and dNTP substrates complementary to the template base. In the enzyme active site, hydrogen bonds can form between substrate and template bases in accordance with Watson-Crick rules to guide
480
ECHOLS & GOODMAN
this process. However, non-Watson-Crick base pairs may also form; the task of the base insertion pathway is to discriminate against these noncanonical base pairs. There are several possible control points in the nucleotide insertion path way that are likely to be used to discriminate between Watson-Crick and non-Watson-Crick base pairs. Discrimination might initially occur in the dNTP substrate-binding stage of thc reaction. The length of time that a
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
substrate remains bound on a polymerase-DNA complex may be determined by nonspecific nucleotide binding at the polymerase triphosphate binding site, hydrogen bonding between substrate and template bases, and nearest neighbor base-stacking interactions. Since base-pairing (H-bonding and base-stacking) interactions stabilize correct base pairs preferentially over incorrect base pairs, dissociation from the polymerase-DNA complex might occur more rapidly for a mispaired substrate
(4--6). A second stage of discrimination
could involve an enzyme conformational change strongly favoring correct over incorrect substrates (7-9). Finally, the rate of phosphodiester bond formation with release of pyrophosphate could occur more rapidly for correct dNTPs (7-9). Different steps in the insertion pathway could be used to a greater or lesser extent by individual polymerases to optimize insertion fidel ity. The recent introduction of enzyme kinetic approaches has sharpened the analysis of replication fidelity. As described below, pre-steady-state kinetic analysis with pol I of Escherichia coli has provided quantitative estimates of the discrimination for each step in the nucleotide insertion pathway
(7-9).
Steady-state kinetic methods have provided a simple and rapid approach to measurements of overall insertion fidelity (l 0-16).
Structural Aspects to Base Selection by DNA Polymerases In their original papers on DNA structure, Watson & Crick pointed out that errors in DNA replication were expected from the properties of base pairs (17,
18). These authors pointed out that A·C and G· T transition mispairs could occur as Watson-Crick structures consisting of two and three H-bonds respec tively, provided that either of the mispaired bases was present as a disfavored imino (A or C) or enol (G or T) tautomer. In addition to tautomeric base shifts, disfavored tautomer models for transversions require rotating the base relative to sugar from an ami to syn conformation (19). Two key points are implied by tautomer shift schemes: geometrical constraints imposed by a polymerase active site strongly favor Watson-Crick over non-Watson-Crick structures; and two H-bonds are better than one for mismatch stabilization
( 1 7-20). During the past several years, the structure of aberrant base pairs in duplex DNA oligonucleotides has been analyzed by X-ray crystallography (21-26) and nuclear magnetic resonance (NMR) (27-34). Experiments have been
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FIDELITY MECHANISMS IN DNA REPLICATION
481
performed Illsing natural bases (2 1 -23, 25-30, 32, 33), base analogues (24, 34-37), and abasic (apurinic/apyrimidinic) lesions (38-40). These studies have revealed that base mispairs are generally stabilized by more than a single H-bond; in none of the studies, however, werc disfavored tautomeric struc tures observed. Instead, the source of additional H-bonding between bases came from solvent-base interactions leading to either protonated or ionized base mispairs, or from wobble structures. A protonated A'C mispair (33) was observed to be in a wobble conformation (21, 32, 33); ionized Watson-Crick pairs in equilibrium with ncutral wobble structures were found for the case of fluorouracil or bromorouracil paired with guanine (36, 37); G'T pairs were detected as wobble structures (2 1 , 3 1 ); and G·A pairs were observed in an anti-anti glycosidic configuration (29, 3 1 ), as G'A(syn) (23), or as G(syn)'A (25). For DNA containing an abasic lesion, A located opposite the lesion was found to stack within the double helix with no discernible DNA distortion (38, 39); G and T revealed both intra- and extrahelical components; C appeared to be exclusively extrahelical (40). When attempting to relate structural data on base mispairs to their forma tion by polymerases, it is important to keep in mind that the most stable mispaired structures derived from X-ray crystallography and NMR or by an analysis of melting temperatures are not necessarily the misinsertions made most efficiently by the enzyme. For the base analogue 2-aminopurine (2AP), DNA containing 2AP'A wobble pairs (35) was found to be more stable (4 1 ) than that containing 2AP'C protonated Watson-Crick pairs (34) . Yet polymerase forms 2AP'C pairs much more efficiently than 2AP'A (42). The same discrepancy appears to be true for the base xanthine (X); insertion of C opposite X occurs much more efficiently than insertion of G opposite X, even though polymers containing X'G are more stable than those with X'C (43). The key point may be that polymerases strongly prefer to insert nucleotides in the configuration closest to a canonical Watson-Crick structurc, even when the structure may be aberrant. Yet, in product DNA, a well-stacked wobble pair may result in a more stable structure than a poorly stacked base pair approximating Watson-Crick geometry more closely. From energy considerations, the geometrical and electrostatic properties of the polymerase active site are likely to exert a strong influence on nucleotide insertion specificities. Based on measurements of melting temperature differ ences between matched and mismatched base pairs in aqueous solution, free energy differences are estimated to be in the range of 0.2 to 0.4 kcal/mol for terminal pairs (44) and 1 to 3 kcaVmol for internal pairs (45). Assuming that the energetic properties of a terminal mismatch approximate those of an incoming dNTP, the calculated discrimination between right and wrong base pairs would be only in the tenfold range (based on the calculated differential equilibrium dissociation constants for correct vs incorrect base pairs). DNA polymerase insertion accuracies have been measured in the range of 103 to 105
482
ECHOLS & GOODMAN
in vitro , requiring aaG values to be much larger than those measured in water. Petruska et al (44) have pointed out that the free energy differences between right and wrong base pairs in aqueous solution are small because of "entropy enthalpy" compensation; ��SO is proportional to �aHo in aqueous medium. Thus, relatively large aaHo values are nearly cancelled by large TaaSo values, resulting in small aaGo values (aaGo aaW - TaaSO). Entropy enthalpy compensation in aqueous solution is observed not only for DNA molecules (44), but also for proteins (46, 47), protein-DNA complexes (48) , and drug-DNA complexes (49). A possible means for a polymerase to exploit large enthalpy differences between right and wrong base pairs is to suppress differences in entropy (44) . The geometric properties of the polymerase active site might provide this entropy suppression. Further amplification of enthalpy differences between right and wrong base pairs might be achieved by partial exclusion of water from the polymerase active site (50). A combination of enthalpy amplification and entropy suppression could markedly amplify the free energy differential between correct and incorrect base pairs.
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=
Polymerase Insertion Errors Insertion of mismatched bases by DNA polymerases occur infrequently . Typically, error frequencies in vitro occur at the 10-3 to 10-5 level, although substantial variation has been observed dependent on the DNA polymerase, method of assay, and specific site investigated. The most common mispairs generally involve G pairing with T, with observed frequencies in a range of 10-2 to 10-4, and the least common mispairs involve pyr'pyr mispairs at frequencies of about 10-4 to 10-5 or less (13, 16, 45, 51-56). The wide variations in error range for a given mismatch can be attributed to several factors. First, DNA polymerases differ in their base misinsertion specificities and intrinsic fidelity. Second, there are specific template-primer sites where base insertion errors are found to occur at anomalously high and low frequencies (hot and cold spots , respectively) . In an effort to focus on how DNA polymerase chooses the correct base, we have emphasized single base insertion errors in this review . It is important to note , however, that misalignment of the template-primer constitutes a major source of mutation in vivo and in vitro (see Kunkel & Bebenek (57) for an excellent review of recent work). Misalignment errors can yield either frameshift mutations (58-6 1 ) or substantial rearrangements (62). Kunkel and collaborators have recently provided an important new insight about misalign ment errors-that transient misalignment can yield base substitution muta tions (59, 63, 64) (Figure I). Because misalignment errors are dependent on DNA sequence , a mutational hot spot may be generated (58 , 65) . The majority of hot and cold mutational sites, however, probably cannot be explained solely in terms of misalignment errors (52 , 66, 67).
FIDELITY MECHANISMS IN DNA REPLICATION
dTTP
" --- T-�
+
---II-T-T-II-C
C, Base Substitution
S, Dislocation
A. Misinsertion
483
dTTP
!-�
us.
+
II T-II-C "
-----
!-�-T
II-T-T-II-C
T
or
D, Frame Shift
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
dTTP dGTP "
Figure 1
!-�
++
R T-R-C " T
-----
5'
"
T-II-T-6
it" j-it-i: T
Mutagenic misalignment of the primer-template for DNA replication. Base substitu
tion mutatiom. are typically caused by mispairing of an incorrect dNTP opposite a template base
(A), A transient misalignment ("dislocation") of the primer-template (8), however, can yield base substitution (C) or frame-shift mutations (D) even though the base insertion event involves a Watson-Crick pair.
From data obtained recently with E, coli strains defective in mismatch correction and editing , a rough estimate is possible of the frequency and type of base insertion error produced by pol III holoenzyme in vivo. As described in more detail below, the mutD5 (dnaQ) mutation confers a severe defect on the editing exonuclease (E subunit) of pol III holoenzyme (68-70) . Recent work has indicated strongly that E, coli carrying the mutD5 mutation are also phenotypically defective in mismatch repair, probably because the mismatch repair pathway is saturated at high mutation rates (65 , 71, 72), Because mutD5 can provide an increase in mutation rate up to 105 (65 , 73 , 74) , the maximum base insertion error frequency by DNA pol III holoenzyme is probably in the 1 0-5 range (10-5 out of 1 0-10 overall); this number is within an order of magnitude of that observed by the a polymerase subunit of pol III holoenzyme in vitro (5 1 ), Of the base insertion mutations found in the mutD5 strain , transition mutations predominate (90%) (65), These mutations would arise from G-T and A'C wobble pairings . Transversion mutations that would arise from. pyf'pyr mispairs are very rare. As described below, the class of transversion mutations arising from G·A mispairs would be relatively high, but these errors are prevented by a special editing system, mutT . Thus , G'T, A-C , and G'A mispairings are probably the most frequent allowed by the polymerase site of pol III in vivo, These misinscrtion errors are also probably the most frequent in vitro for the a-subunit of pol III holoenzyme (51),
Fidelity Assays and Major Conclusions Four main assays are presently used to analyze the fidelity of replication by purified polymerases in vitro or the process of genome replication in vivo.
484
ECHOLS & GOODMAN
MIS INCORPORATION
AND
PROOFREADING
IN
VITRO
MEASURED
BY
A
In the first misincorporation demon stration in 1962, Trautner et al showed with E. coli DNA polymerase I that G was misinserted opposite bromouracil on d(ABrU) but not opposite T on d(AT) (75). Subsequently, Hall & Lehman used a similar assay to show that a mutator mutant of T4 polymerase misincorporated T opposite C more fre quently than wild type (76). The incorporation assay was generalized to include a measurement of deoxynucleotide turnover, the conversion of newly inserted nucleotides into acid-soluble dNMPs by polymerase-associated exo nuclease (77, 78). In the "standard" polymerase assay, measurements are generally carried out with both matched and mismatched dNTPs present in the reaction simultaneously, so that the two can compete directly for insertion into DNA. The misinsertion ratio,lin" is defined as the ratio of wrong to right substrates incorporated into DNA, normalized to equimolar substrate con centrations. Fidelity is defined to be the reciprocal of the misinsertion ratio, F l/hns. The misinsertion ratio can be determined by tagging right and wrong dNTPs with different radioactive labels and measuring their relative in corporation into DNA and turnover as dNMPs, where nucleotide insertion is given by the sum of incorporation plus turnover (77-S0); alternatively, com petition for incorporation can be measured between an unlabeled and labeled substrate ( 1 0) . The insertion efficiencies for mismatched nucleotides are generally between three to five orders of magnitude less than for correctly matched nucleotides. Thus, to detect incorporation of mismatched bases, it is frequently necessary to provide a substantial pool bias by increasing the relative concentration of mismatched to matched dNTP. The measurement of insertion of radioactive deoxynucleotide substrates into DNA and subsequcnt proofreading of newly inserted substrates by exonu clease continues to serve as a standard "workhorse" assay to address a wide variety of important questions concerning fidelity of DNA synthesis. Fidelity measurements have been made for a large number of polymerases by using the "standard" assay to estimate average fidelity values and by applying more recent assays to determine fidelity at individual template sites (for previous reviews, see Refs. 45, 57, SI). Similar studies have been carried out to determine base mispairing properties of nucleotide analogues and the effects of mutagens and carcinogens on polymerase fidelity (11, 12, 15, 42, 75, 80, 82-1 02).
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"STANDARD" POL YMERASE ASSAY
=
REVERSION,
TRANSFECTION,
AND
SHUTTLE
VECTOR
SYSTEMS
USING
An understanding of replication fidelity requires data on the mutation process in vivo. The importance of the DNA polymerase in providing fidelity was initially established by forward mutational and genetic reversion studies using a dispensable phage T4 gene (e.g. rII locus) as a marker ( 1 03-1 07). An assay measuring spontaneous or mutagen-induced REPORTER GENES
ADELITY MECHANISMS IN DNA REPLICATION
reversion of nonsense mutations in the lad gene of E. coli was introduced by Miller and coworkers to determine the mutational spectrum associated with genomic duplication in vivo (108, 1 09). Recently, a series of lacZ mutations were defined that allow rapid detection by reversion of all base substitution mutations. ( 1 09a, 1 09b). Another general protocol that has been used extensively involves expres sion of
Further
Quick links to online content Annu. Rev. Biochem. Copyright ©
199/
1991. 60:477-511
by Annual Reviews Inc. All rights reserved
FIDELITY MECHANISMS Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
IN Dl'IA REPLICATION Harrison Echols Department of Molecular and Cell Biology, University of California, Berkeley, California
94720
Myron F. Goodman Department of Biological Science, University of Southern California, Los Angeles,
California 90089-1340 KEY WORDS:
DNA polymerases, mutation mechanisms, DNA damage, editing
by DNA
polymerases.
CONTENTS PERSPECTIVES AND SUMMARY ............................................................. .
478
BASE INSERTION SELECTIVITY BY DNA POLYMERASES ......................... .
479 479
The Base 1''lSertion Pathway ................................................................... . Structural Aspects to Base Selection by DNA Polymerases ............................. . Polymerase Insertion Errors ................................................................... . Fidelity Assays and Major Conclusions ..................................................... . Error Avoidance in Base Insertion----General Conclusions ..... ........................ . EDITING BY DNA POLYMERASES........................................................... .
Exonucleolytic Editing by Prokaryotic Polymerases ...................................... . Editing in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetic Amplification of Editing: Inefficient Extension from a Mismatched 3'-Terminus.......................................................... . Error Avoidance in Editing----General Conclusions ....................................... . Possible Additional Editing Mechanisms: the MutT Pathway ........................... . MUTATIONS GENERATED BY DAMAGE TO DNA ..................................... .
Response of DNA Polymerase to DNA Lesions ............................................ . The SOS R"sponse of Escherichia coli ....................................................... Multiple Roles of RecA in the SOS Response .............................................. . Some Biological Comments .................................................................... .
480 482 483 488 491 491 492 493 494 496 497 497 499 500 505
477 0066-4 1 54/91/0701 -0477$02.00
478
ECHOLS & GOODMAN
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
PERSPECTIVES AND SUMMARY The survival of an organism requires precise duplication of the genome. Error frequencies are typically only 10-9 to 10 10 per base replicated. The bio chemical problem posed by this demand for faithful duplication is ex ceptionally complex because the correct (Watson-Crick) base pairs do not exhibit large structural or energetic differences from some incorrect base pairs (e. g . G·T vs A·T) . For the well-defined prokaryotic replication systems , the requirement for a low mutation rate is achieved by the sequential operation of three fidelity mechanisms: (a) selection of the correct deoxynucleoside triphosphate (dNTP) substrate in the polymerization reaction (base selection); (b) exonucleolytic removal of an incorrectly inserted deoxynucleoside monophosphate (dNMP) from the end of the growing chain (editing); (c) postreplicative excision of an incorrectly inserted deoxynucleoside monophosphate after the polymerase has extended the DNA chain (mismatch repair). Each of these mechanisms exhibits an impressive selectivity for excluding noncanonical base pairs; the sequential application of all three fidelity operations yields precisely duplicated DNA. For eukaryotic replica tion systems, only base selection has so far been clearly defined as a general fidelity mechanism. However, most cellular and viral eukaryotic polymerases exhibit exonucleolytic editing activities , and there has been excellent recent evidence for mismatch correction pathways. Thus the prokaryotic solutions described here are likely to be general . In this review we consider in detail only the two coupled fidelity mech anisms that involve the direct participation of the DNA polymerase-base selection and editing. Excellent recent reviews have appeared on mismatch repair (1-3). We focus on the fundamental biochemical question: how does a DNA polymerase distinguish a correct from an incorrect base pair with such astounding precision? Recent structural and kinetic data indicate that polymerases achieve selectivity by mechanisms that differ in detail . However, from the available evidence, we believe that all polymerases follow general fidelity principles for base selection and editing. For base selection, the most likely discrimination principle is an exquisite demand for the precise geometry of the Watson-Crick base pair. The most remarkable structural property of the A'T and G'C base pairs is their complete geometric equivalence as subunits of the double helix; all other non-Watson Crick base pairs known to occur (e. g. the G·T "wobble" pair) have altered geometry . We presume that the active site of the polymerase exerts transition state selectivity for the identical geometry (CI' distances and bond angles) of the Watson-Crick base pairs. As a consequence, the correct dNTP is used 104 or 105 times as efficiently as an incorrect dNTP. This demand for Watson Crick geometry at the transition state might be manifested either in more rapid dissociation of incorrect dNTP, or slower phosphodiester bond formation for
FIDELITY MECHANISMS IN DNA REPLICATION
479
misoriented bases , or most likely both. The active site of a polymerase probably evolved to catalyze phosphodiester bond formation with either A, T ,
G, o r C i n the dNTP site, with frequency dependent o n pairing energetics between substrate and template. Thus a geometric selection mechanism could be achieved by fine-tuning the allowed template-substrate configurations . For exonucleolytic editing , the most likely discrimination mechanism is the "melting capacity" of the mispaired DNA; a misaligned base at the 3' end will be more often in a single-strand configuration. This mechanism is an easy
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
evolutionary transition from a free single-strand exonuclease. However, the degree of discrimination against a mispair is likely to be relatively small, especially with respect to the more thermally sensitive A'T correct pairs. Editing bec:omes an efficient process, with discrimination of 102 or more , because of kinetic amplification mechanisms. In the most clearly defined mechanism, the next base addition reaction following a misinsertion is slow compared to base addition following a correct insertion. Thus the exonuclease has longer to act on the mispair, amplifying the editing discrimination. Certain DNA lesions inhibit DNA replication. The damage site is presum ably suffici(!ntly distorted that the geometric recognition mechanism does not allow effective base insertion. In addition, the editing exonuclease will preferentiaHy remove even a "correctly" inserted base because the resultant distorted base pair looks like a mismatch. The rescue of such a stalled polymerase requires a complex cellular response. In the last section of this review, we consider briefly the induced "SOS response" to replication blocking lesions. The SOS response provides for a highly mutagenic pathway of "translesion" DNA replication, dependent on induced synthesis of the mutation proteins UmuC and UmuD, followed by regulated processing of UmuD to its active form, UmuD'. An interesting point is that mutations are introduced by an induced and highly regulated pathway; SOS mutagenesis is not a passive response to DNA damage. The existence of an induced mutation pathway might seem puzzling in view of our opening statement that the mutation rate must be kept low. However, mutation is the source of genetic variation, and a regulated loss of replication fidelity might serve to accelerate evolutionary adaptation in an endangered population. Thus the fidelity of DNA replication may be subject to environmental regulation.
BASE INSERTION SELECTIVITY BY DNA POLYMERASES
The Base Insertion Pathway The role of a D NA polymerase is to catalyze the formation of phosphodiester bonds between primer 3' -temlini and dNTP substrates complementary to the template base. In the enzyme active site, hydrogen bonds can form between substrate and template bases in accordance with Watson-Crick rules to guide
480
ECHOLS & GOODMAN
this process. However, non-Watson-Crick base pairs may also form; the task of the base insertion pathway is to discriminate against these noncanonical base pairs. There are several possible control points in the nucleotide insertion path way that are likely to be used to discriminate between Watson-Crick and non-Watson-Crick base pairs. Discrimination might initially occur in the dNTP substrate-binding stage of thc reaction. The length of time that a
Annu. Rev. Biochem. 1991.60:477-511. Downloaded from www.annualreviews.org by New York University - Bobst Library on 10/19/14. For personal use only.
substrate remains bound on a polymerase-DNA complex may be determined by nonspecific nucleotide binding at the polymerase triphosphate binding site, hydrogen bonding between substrate and template bases, and nearest neighbor base-stacking interactions. Since base-pairing (H-bonding and base-stacking) interactions stabilize correct base pairs preferentially over incorrect base pairs, dissociation from the polymerase-DNA complex might occur more rapidly for a mispaired substrate
(4--6). A second stage of discrimination
could involve an enzyme conformational change strongly favoring correct over incorrect substrates (7-9). Finally, the rate of phosphodiester bond formation with release of pyrophosphate could occur more rapidly for correct dNTPs (7-9). Different steps in the insertion pathway could be used to a greater or lesser extent by individual polymerases to optimize insertion fidel ity. The recent introduction of enzyme kinetic approaches has sharpened the analysis of replication fidelity. As described below, pre-steady-state kinetic analysis with pol I of Escherichia coli has provided quantitative estimates of the discrimination for each step in the nucleotide insertion pathway
(7-9).
Steady-state kinetic methods have provided a simple and rapid approach to measurements of overall insertion fidelity (l 0-16).
Structural Aspects to Base Selection by DNA Polymerases In their original papers on DNA structure, Watson & Crick pointed out that errors in DNA replication were expected from the properties of base pairs (17,
18). These authors pointed out that A·C and G· T transition mispairs could occur as Watson-Crick structures consisting of two and three H-bonds respec tively, provided that either of the mispaired bases was present as a disfavored imino (A or C) or enol (G or T) tautomer. In addition to tautomeric base shifts, disfavored tautomer models for transversions require rotating the base relative to sugar from an ami to syn conformation (19). Two key points are implied by tautomer shift schemes: geometrical constraints imposed by a polymerase active site strongly favor Watson-Crick over non-Watson-Crick structures; and two H-bonds are better than one for mismatch stabilization
( 1 7-20). During the past several years, the structure of aberrant base pairs in duplex DNA oligonucleotides has been analyzed by X-ray crystallography (21-26) and nuclear magnetic resonance (NMR) (27-34). Experiments have been
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FIDELITY MECHANISMS IN DNA REPLICATION
481
performed Illsing natural bases (2 1 -23, 25-30, 32, 33), base analogues (24, 34-37), and abasic (apurinic/apyrimidinic) lesions (38-40). These studies have revealed that base mispairs are generally stabilized by more than a single H-bond; in none of the studies, however, werc disfavored tautomeric struc tures observed. Instead, the source of additional H-bonding between bases came from solvent-base interactions leading to either protonated or ionized base mispairs, or from wobble structures. A protonated A'C mispair (33) was observed to be in a wobble conformation (21, 32, 33); ionized Watson-Crick pairs in equilibrium with ncutral wobble structures were found for the case of fluorouracil or bromorouracil paired with guanine (36, 37); G'T pairs were detected as wobble structures (2 1 , 3 1 ); and G·A pairs were observed in an anti-anti glycosidic configuration (29, 3 1 ), as G'A(syn) (23), or as G(syn)'A (25). For DNA containing an abasic lesion, A located opposite the lesion was found to stack within the double helix with no discernible DNA distortion (38, 39); G and T revealed both intra- and extrahelical components; C appeared to be exclusively extrahelical (40). When attempting to relate structural data on base mispairs to their forma tion by polymerases, it is important to keep in mind that the most stable mispaired structures derived from X-ray crystallography and NMR or by an analysis of melting temperatures are not necessarily the misinsertions made most efficiently by the enzyme. For the base analogue 2-aminopurine (2AP), DNA containing 2AP'A wobble pairs (35) was found to be more stable (4 1 ) than that containing 2AP'C protonated Watson-Crick pairs (34) . Yet polymerase forms 2AP'C pairs much more efficiently than 2AP'A (42). The same discrepancy appears to be true for the base xanthine (X); insertion of C opposite X occurs much more efficiently than insertion of G opposite X, even though polymers containing X'G are more stable than those with X'C (43). The key point may be that polymerases strongly prefer to insert nucleotides in the configuration closest to a canonical Watson-Crick structurc, even when the structure may be aberrant. Yet, in product DNA, a well-stacked wobble pair may result in a more stable structure than a poorly stacked base pair approximating Watson-Crick geometry more closely. From energy considerations, the geometrical and electrostatic properties of the polymerase active site are likely to exert a strong influence on nucleotide insertion specificities. Based on measurements of melting temperature differ ences between matched and mismatched base pairs in aqueous solution, free energy differences are estimated to be in the range of 0.2 to 0.4 kcal/mol for terminal pairs (44) and 1 to 3 kcaVmol for internal pairs (45). Assuming that the energetic properties of a terminal mismatch approximate those of an incoming dNTP, the calculated discrimination between right and wrong base pairs would be only in the tenfold range (based on the calculated differential equilibrium dissociation constants for correct vs incorrect base pairs). DNA polymerase insertion accuracies have been measured in the range of 103 to 105
482
ECHOLS & GOODMAN
in vitro , requiring aaG values to be much larger than those measured in water. Petruska et al (44) have pointed out that the free energy differences between right and wrong base pairs in aqueous solution are small because of "entropy enthalpy" compensation; ��SO is proportional to �aHo in aqueous medium. Thus, relatively large aaHo values are nearly cancelled by large TaaSo values, resulting in small aaGo values (aaGo aaW - TaaSO). Entropy enthalpy compensation in aqueous solution is observed not only for DNA molecules (44), but also for proteins (46, 47), protein-DNA complexes (48) , and drug-DNA complexes (49). A possible means for a polymerase to exploit large enthalpy differences between right and wrong base pairs is to suppress differences in entropy (44) . The geometric properties of the polymerase active site might provide this entropy suppression. Further amplification of enthalpy differences between right and wrong base pairs might be achieved by partial exclusion of water from the polymerase active site (50). A combination of enthalpy amplification and entropy suppression could markedly amplify the free energy differential between correct and incorrect base pairs.
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=
Polymerase Insertion Errors Insertion of mismatched bases by DNA polymerases occur infrequently . Typically, error frequencies in vitro occur at the 10-3 to 10-5 level, although substantial variation has been observed dependent on the DNA polymerase, method of assay, and specific site investigated. The most common mispairs generally involve G pairing with T, with observed frequencies in a range of 10-2 to 10-4, and the least common mispairs involve pyr'pyr mispairs at frequencies of about 10-4 to 10-5 or less (13, 16, 45, 51-56). The wide variations in error range for a given mismatch can be attributed to several factors. First, DNA polymerases differ in their base misinsertion specificities and intrinsic fidelity. Second, there are specific template-primer sites where base insertion errors are found to occur at anomalously high and low frequencies (hot and cold spots , respectively) . In an effort to focus on how DNA polymerase chooses the correct base, we have emphasized single base insertion errors in this review . It is important to note , however, that misalignment of the template-primer constitutes a major source of mutation in vivo and in vitro (see Kunkel & Bebenek (57) for an excellent review of recent work). Misalignment errors can yield either frameshift mutations (58-6 1 ) or substantial rearrangements (62). Kunkel and collaborators have recently provided an important new insight about misalign ment errors-that transient misalignment can yield base substitution muta tions (59, 63, 64) (Figure I). Because misalignment errors are dependent on DNA sequence , a mutational hot spot may be generated (58 , 65) . The majority of hot and cold mutational sites, however, probably cannot be explained solely in terms of misalignment errors (52 , 66, 67).
FIDELITY MECHANISMS IN DNA REPLICATION
dTTP
" --- T-�
+
---II-T-T-II-C
C, Base Substitution
S, Dislocation
A. Misinsertion
483
dTTP
!-�
us.
+
II T-II-C "
-----
!-�-T
II-T-T-II-C
T
or
D, Frame Shift
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dTTP dGTP "
Figure 1
!-�
++
R T-R-C " T
-----
5'
"
T-II-T-6
it" j-it-i: T
Mutagenic misalignment of the primer-template for DNA replication. Base substitu
tion mutatiom. are typically caused by mispairing of an incorrect dNTP opposite a template base
(A), A transient misalignment ("dislocation") of the primer-template (8), however, can yield base substitution (C) or frame-shift mutations (D) even though the base insertion event involves a Watson-Crick pair.
From data obtained recently with E, coli strains defective in mismatch correction and editing , a rough estimate is possible of the frequency and type of base insertion error produced by pol III holoenzyme in vivo. As described in more detail below, the mutD5 (dnaQ) mutation confers a severe defect on the editing exonuclease (E subunit) of pol III holoenzyme (68-70) . Recent work has indicated strongly that E, coli carrying the mutD5 mutation are also phenotypically defective in mismatch repair, probably because the mismatch repair pathway is saturated at high mutation rates (65 , 71, 72), Because mutD5 can provide an increase in mutation rate up to 105 (65 , 73 , 74) , the maximum base insertion error frequency by DNA pol III holoenzyme is probably in the 1 0-5 range (10-5 out of 1 0-10 overall); this number is within an order of magnitude of that observed by the a polymerase subunit of pol III holoenzyme in vitro (5 1 ), Of the base insertion mutations found in the mutD5 strain , transition mutations predominate (90%) (65), These mutations would arise from G-T and A'C wobble pairings . Transversion mutations that would arise from. pyf'pyr mispairs are very rare. As described below, the class of transversion mutations arising from G·A mispairs would be relatively high, but these errors are prevented by a special editing system, mutT . Thus , G'T, A-C , and G'A mispairings are probably the most frequent allowed by the polymerase site of pol III in vivo, These misinscrtion errors are also probably the most frequent in vitro for the a-subunit of pol III holoenzyme (51),
Fidelity Assays and Major Conclusions Four main assays are presently used to analyze the fidelity of replication by purified polymerases in vitro or the process of genome replication in vivo.
484
ECHOLS & GOODMAN
MIS INCORPORATION
AND
PROOFREADING
IN
VITRO
MEASURED
BY
A
In the first misincorporation demon stration in 1962, Trautner et al showed with E. coli DNA polymerase I that G was misinserted opposite bromouracil on d(ABrU) but not opposite T on d(AT) (75). Subsequently, Hall & Lehman used a similar assay to show that a mutator mutant of T4 polymerase misincorporated T opposite C more fre quently than wild type (76). The incorporation assay was generalized to include a measurement of deoxynucleotide turnover, the conversion of newly inserted nucleotides into acid-soluble dNMPs by polymerase-associated exo nuclease (77, 78). In the "standard" polymerase assay, measurements are generally carried out with both matched and mismatched dNTPs present in the reaction simultaneously, so that the two can compete directly for insertion into DNA. The misinsertion ratio,lin" is defined as the ratio of wrong to right substrates incorporated into DNA, normalized to equimolar substrate con centrations. Fidelity is defined to be the reciprocal of the misinsertion ratio, F l/hns. The misinsertion ratio can be determined by tagging right and wrong dNTPs with different radioactive labels and measuring their relative in corporation into DNA and turnover as dNMPs, where nucleotide insertion is given by the sum of incorporation plus turnover (77-S0); alternatively, com petition for incorporation can be measured between an unlabeled and labeled substrate ( 1 0) . The insertion efficiencies for mismatched nucleotides are generally between three to five orders of magnitude less than for correctly matched nucleotides. Thus, to detect incorporation of mismatched bases, it is frequently necessary to provide a substantial pool bias by increasing the relative concentration of mismatched to matched dNTP. The measurement of insertion of radioactive deoxynucleotide substrates into DNA and subsequcnt proofreading of newly inserted substrates by exonu clease continues to serve as a standard "workhorse" assay to address a wide variety of important questions concerning fidelity of DNA synthesis. Fidelity measurements have been made for a large number of polymerases by using the "standard" assay to estimate average fidelity values and by applying more recent assays to determine fidelity at individual template sites (for previous reviews, see Refs. 45, 57, SI). Similar studies have been carried out to determine base mispairing properties of nucleotide analogues and the effects of mutagens and carcinogens on polymerase fidelity (11, 12, 15, 42, 75, 80, 82-1 02).
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"STANDARD" POL YMERASE ASSAY
=
REVERSION,
TRANSFECTION,
AND
SHUTTLE
VECTOR
SYSTEMS
USING
An understanding of replication fidelity requires data on the mutation process in vivo. The importance of the DNA polymerase in providing fidelity was initially established by forward mutational and genetic reversion studies using a dispensable phage T4 gene (e.g. rII locus) as a marker ( 1 03-1 07). An assay measuring spontaneous or mutagen-induced REPORTER GENES
ADELITY MECHANISMS IN DNA REPLICATION
reversion of nonsense mutations in the lad gene of E. coli was introduced by Miller and coworkers to determine the mutational spectrum associated with genomic duplication in vivo (108, 1 09). Recently, a series of lacZ mutations were defined that allow rapid detection by reversion of all base substitution mutations. ( 1 09a, 1 09b). Another general protocol that has been used extensively involves expres sion of
