Biochimie ( 199 i ) 73,269-276
© SociEt6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris
269
Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro WD Heyer*, AW Johnson, DN Norris, D Tishkoff, RD Kolodner** Department of Biological Chemistry and Molectdar Pharmacology, Harvard Medical School," Division ~" Cell and Molecldar Biology, Dana-Father Cancer Institute. 44 Binne v Street, Boston. M4 02 i 15 USA
(Received 29 October 1990; accepted 25 January 1991 )
Summary - - RecA-like activities that can form hybrid DNA ill vitro have been identified in a wide variety of organisms. We have previously described the strand exchange protein 1 (SEPI) from the yeast Saccharomyces cerevisiae that can form hybrid DNA in vitro. Purified as an Mr 132 000 polypeptide, recent molecular and immunological studies have now shown that the native form is an Mr 175 000 polypeptide containing strand exchange activity. The gene encod;ng SEPI has been cloned and sequenced. The primary sequence failed to reveal any significant sequence homology to other sequences in data base searches, hi vivo SEPI was found to be essential for normal meiosis as cells containing a homozygous insertion mutation in the SEPI gene failed to sporulate. In order to identify additional factors that are involved in hybrid DNA formation in S cerevisiae, we used an in vitro stimulation assay to identify proteins that reconstitute strand exchange activity in reactions containing limiting amounts of SEPI. We have identified two proteins that functionally interact with SEPI. First, an Mr 34 000 single-stranded DNA binding protein stimulated the reaction by lowering the requirement for SEPI about 3--4 fold. This protein is a fragment of the large subunit of a hetero-trimeric complex called yRP-A (yRF-A) which is thought to be the functional eukaryotic equivalent of single-stranded DNA binding proteins in prokaryotes. The gene encoding this protein (RPAI) is essential for growth. Second, an Mr 33 000 polypeptide, termed Stimulatory Factor 1 (SF1), dramatically stimulated the SEPI catalyzed reaction by lowering the requirement for SEPI about 300 fold. This protein specifically stimulated SEPI in a way that is quantitatively and qualitatively different from previously found stimulation of strand exchange proteins. S cerevisiae / recombination / strand exchange / hybrid DNA
Introduction
Genetic recombination is a fundamental cellular process which is implicated in a) the generation of genetic diversity, b) the repair of DNA damage, c) the homologous alignment of chromosomes before meiotic cell division, and d) the generation of genomic alterations that lead to changes in gene expression. Based mainly on fungal tetrad data, molecular models have been developed to account for the observed consequences of recombination events [1-3]. C o m m o n to all these models is the central role of the hybrid DNA intermediate. In fungi, hybrid D N A can be directly traced in the phenomenon called PMS (Post Meiotic S_egregation) where a DNA mismatch originating from the hybrid DNA has escaped mismatch repair, resulting in an impure *Present address: Institute of General Microbiology, BaltzerStr.4, CH-3012 Bern, Switzerland **Correspondence and reprints
meiotic product that will segregate the characters upon the first mitosis usmg proper genetical techniques. Thus, PMS can be a direct measure of hybrid D N A formation giving firm evidence for this intermediate [4]. The identification of the recA gene in Escherichia colt [5] and the functional analysis of the recA gene product further established the pivotal importance of the hybrid D N A intermediate in homologous recombination [6, 7]. The mechanics of hybrid DNA formation in vitro by the recA protein have been studied in great detail. Often the model reaction shown in figure 1 has been used. The assay makes use of simple DNA substrates with easy detection of the reaction products by gel electrophoresis. The confirmation by electron microscopy of the structure of the joint molecules as shown in figure 1 is essential. The ease of the strand exchange assay has prompted the search for r e c A - l i k e activities that can form hybrid D N A in vitro in m a n y organisms. Such activities have been detected in a wide variety
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S cerevisiae displays high levels of homologous recombination and a wealth of data from genetical studies exists [4]. Together with the experimental possibilities of reverse genetics, yeasts are the ideal eukaryotic model system to study homologous recombination. By focussing on the formation of the central intermediate, hybrid DNA, we have used the assay shown in figure 1 to purify the strand exchange protein, SEP 1 [9]. To identify additional proteins involved in hybrid DNA formation, we have used an in vitro stimulation assay using limiting amounts of SEPI assaying for reconstitution of strand exchange activity. We found two proteins that stimulated SEP1 in vitro. First, a single-stranded DNA binding protein [a fragment of yRP-A (yRF-A)] and second, a novel protein called Stimulatory Factor 1 (SF 1).
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Fig 1. Hybrid DNA assay: the strand exchange reaction. Linear double-stranded DNA (MI3 replicative form) and circular single-stranded DNA (M13 viral DNA) are the substrates. The products of a strand exchange reaction are collectively called joint molecules. Electron microscopic analysis can easily distinguish between ~- and s-forms and the end product, open circular DNA. The region of hybrid
Using the assay illustrated in figure 1 we have previously purified the strand exchange protein, SEP 1, from mitotic S cerevisiae cells [9]. The Mr 132 000 form of SEPI formed hybrid DNA in the strand exchange reaction with the polarity shown in figure 1, ie 3' to 5', like E.coli recA protein. SEPI was requ;red in high and stoichiometric amounts in this reaction of 1 M r 132 000 monomer per 12 nucleotides of single-stranded DNA. The formation of joint molecules required the substrates to be homologous. The
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overhang or blunt, had no influence on the efficiency of the reaction. The reaction requirements were rather simple, essentially only the presence of the divalent cation Mg++ was required. In particular, the strand exchange was catalyzed in the absence of any requirement for a high energy cofactor such as ATP. This is in apparent contrast to the situation with recA, which was thought to require ATP hydrolysis to catalyze strand exchange [7]. In addition, SEP1 neither bound ATP, as indicated by photoaffinity-labelling experiments using 8-azido ATP, nor exhibited ATPase activity (WDH and RDK., unpublished results). The absence of an energy requirement does not pose an energetic paradox, as there was no indication for catalytic turnover of the protein. Electron microscopic analysis of the reaction products demonstrated the presence of all three classes of joint molecules as shown in figure 1, namely t~- and t~-structures as well as the endproduct, open circular double-stranded DNA. The physical demonstration of s-structures eliminated the possibility that the formation of joint molecules was generated by the action of a nuclease. The average length of hybrid DNA formed was measured to be 4.1 kbp using a 7.2 kbp substrate.
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of organisms including fungi [Ustilago maydis: 8; S cerevisiae: %12], insects [Drosophila melanogastel': 13-14], mammals [15-21], and 91ants [16, 22]. However, only the fungal activities (cited above), one protein from D melanogaster [23], and one protein from man [24] have been purified to homogeneity. The purified proteins not only allow detailed biochemical characterization of the activity, but also open an avenue to clone the corresponding genes. The gene/protein sequences will provide valuable information on the phylogenetic relationship of these activities and possibly clues about the structurefunction relationship of these proteins. The occurrence of strand exchange activities in this wide range oi" organisms suggests some universal mechanisms in the formation of hybrid DNA. We have taken the yeast S cerevisiae as a model system to study the mechanism(s) of homologous recombination using a biochemical approach [25].
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A number of additional activities are associated with SEP1 that are important for its action in the formation of hybrid DNA in vitro and possibly in vivo [26]. SEPI bound single-stranded DNA as well as double-stranded DNA, but bound double-stranded DNA with lower relative affinity. The binding of SEP1 to the single-stranded DNA rendered the nucleic acid resistant to digestion with nuclease S1 by forming a high molecular weight aggregate that effectively precipitated the DNA apparently excluding access of the nuclease. The formation of these high molecular weight aggregates appeared to be a kinetic intermediate in the formation of joint molecules with strand exchange taking place in these large aggregates. Furthermore, SEP1 catalyzed the renaturation of two complementary single-stranded DNAs, the most simple pairing reaction. The mechanism of the renaturation reaction seemed slightly different to that of the strand exchange reaction in that renaturation was not sensitive to the electrophilic blocking agent N-ethylmaleimide. These characteristics reveal many similarities to the recA protein. RecA binds to single-stranded DNA in a co-operative manner and also renatures complementary single-stranded DNA strands, which can occur even in an ATP independent fashion under certain reaction conditions [7]. In addition, recA causes DNA aggregation which was concluded to be an intermediate in DNA renaturation and strand exchange [27, 28]. Another difference is that SEP1 binds readily to double-stranded DNA, whereas recA has , , to Ua ,U.U, , kOt l ~.-.3.1. .. 1. . d i I U.a~.~ I~U . . . . a. . . k. .i n. .e .t i.c. . . harrier i n h ; n r i ; , ~ , E DNA at neutral pH [291. The gene encoding SEP1 has recently been cloned using degenerate oligonucleotides derived from protein sequencing data, and the DNA sequence has been established (DT and RDK, unpublished results). The sequence revealed an open reading frame with a coding potential for an Mr 175 000 polypeptide showing no significant primary sequence homology to any other protein in data base searches. The full length Mr 175 000 polypeptide has also been overproduced and purified. It was shown to exert strand exchange activity in the strand exchange assay (fig 1) with slightly higher specific activity than the Mr 132 000 form of SEP1 (AWJ and RDK, unpublished results). These data are in agreement with a recent study of Dykstra et al. [ 12] who have also identified the higher molecular weight form of SEP1. Several lines of evidence suggest that the Mr 175 000 polypeptide is the authentic form of SEPI. First, the open reading frame identifying the SEP1 gene has a coding potential for a polypeptide of this size. Second, NH2-terminal amino acid sequencing and proteolysis studies confirmed that in the M, 132 000 form of SEP1 the COOH-terminal - 40 000 kd were . . . . . . . . . . . .
missing. This part of the protein is predicted to be rather unorganized as it shows a high content of proline residues and was found to be protease sensitive. Third, monoclonal antibodies directed against the Mr 132 000 form of SEP1 detected the Mr 175 000 polypeptide in crude cell extracts. Monoclonal antibodies directed against SEP1 readily detected the Mr 175 000 polypeptide in crude cell extracts and identified SEPI as a rather abundant cellular protein. SEPI was found to be present throughout the mitotic cell cycle, during meiosis and was detected in spores. Indirect immunofluorescence light microscopy using anti-SEP1 monoclonal antibodies showed co-localization of SEP1 with the DNA specific fluorescent probe 4,6--diamidino-2-phenyiindole (DAPI) suggesting a nuclear localization of SEP1. (WDH, AWJ, and RDK, unpublished results) The preliminary analysis (DT and RDK, unpublished results) of insertion mutations in the SEP1 gene showed that the gene was not essential for mitotic growth. However the gene was important for mitotic growth as the cells carrying the mutation were clearly growing slower than wild-type. For meiosis, however, SEP1 was essential, since sepl mutants were defective in sporulation resulting in 0-3% ascus formation with 60% spore viability. During meiosis sepl mutant cells underwent an apparently normal DNA synthesis, but were blocked before they proceeded through meiosis I. Cells blocked at this stage could be returned to mitotic -rowth by n l n t i n a t h e m ran , a n n r r a n r ; ~ t a m a A ; . . . . ;*h . . . . significant loss of viability. Preliminary examination of the recombination phenotype of sepl mutant strains showed a very slight reduction (~ 5-fold) in prototroph formation from two his4 hetero-alleles during mitotic growth compared to wild-type. Induction of recombination during meiosis by a factor of 100 to 1000 is a hallmark of yeast genetics. Induction of meiotic prototroph formation from his4 hetero-alleles appeared normal in kinetics but were induced to a greater extent in sepl mutant strains. When mutant cells were examined for the level of prototroph formation after being left at the meiotic block for 24 h after induction of meiosis, they exhibited a 6--10-fold higher meiotic induction than wildtype cells. By this time wild-type cells had already have completed meiosis (ie sporulated) for about 10 h. These preliminary data suggest that the SEPI protein is essential for a successful meiosis, and that the meiotic block in sepl mutants occurs at a stage in meiosis where recombination can take place. An alternative explanation is that sepl mutations block a type of recombination required for meiosis, and that the recombination observed in sepl mutants is promoted by a parallel pathway. --
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Additional factors involved in hybrid DNA formation in vitro A long term biochemical goal in studying genetic recombination is the reconstitution of large parts or of the entire recombination reaction. We have chosen to start with the strand exchange reaction catalyzed by SEPI (fig I). The formation of hybrid DNA is central to genetic recombination, and we envisaged that multiple proteins may function in this reaction in :'ivo. In order to identify additional proteins that r,.re potentially involved in hybrid DNA formation, we have purified proteins that stimulate the SEPI catalyzed reaction in vio'o under conditions where the anaount of SEPI was limiting. The results of this in vio'o stimulation approach are schematically illustrated in figure 2. In practice, strand exchange reactions were set up with amounts of SEPI too low to sustain activity. Column tractions were systematically &ssayed l~or the presence of activities that reconstituted activity. Two proteins were identified and purified by this approach. Mr 34 000 single-stranded DNA binding protein [fragment of yRP-A (yRF-A)] The first protein was identified as an Mr 34 000 polypeptide that showed nucleic binding properties typical for single-stranded DNA binding proteins [30]. The polypeptide had high affinity for single-stranded DNA. Competition experiments showed no or much lower affinity for double-stranded DNA or RNA. The protein. DNA complexes between the Mr 34 000 polypeptide and single-stranded DNA were highly salt stable (elution point from single-stranded DNA cellulose - 1.2 M NaCI). The in vitro functional interaction of the Mr 34 000 polypeptide with SEPI resulted in stimulation of the SEPI catalyzed strand exchange reaction at an optimal stoichiometry of 1 monomer per 18 nucleotides of single-stranded DNA. As shown schematically in figure 2, the amount of SEP1 required to form joint molecules was lowered by 3-4-fold. E coli SSB could not elicit stimulation of SEP1. Conversely, the Mr 34 000 polypeptide stimulated recA as well as E coli SSB did. This is consistent with previous observations that recA can be stimulated by various heterologous singlestranded DNA binding proteins [31]. The Mr 34 000 alone was inert in the strand exchange reaction, only showing an effect when the amount of SEPI was limiting. In this case, both extent and rate of the SEP1 catalyzed reaction were stimulated. The gene encoding the Mr 34 000 polypeptide (RPA',~ was cloned using degenerate oligonucleotide probes and analyzed [32]. The open reading frame identifying the gene showed a coding potential for
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Fig 2. In vitro stimulation of SEP1. Schematic representation of the in vitro stimulation of SEPI by the Mr 34 000 sxingle-stranded DNA binding protein (fragment of the large subunit of yRF-1/RP-A) and by the Mr 33 000 polypeptide stimulatory factor 1 (SFI). Activity indicates the % of joint molecules formed in the strand exchange assay described in figure 1. The log of relative protein concentration is given to illustrate the amount of stimulation mediated by the different proteins. The amount of protein corresponds for SEPI to 550 Mr 132 000 monomer per single-stranded DNA substrate molecule, for SEP1 + RPAI* (Mr 34 000 fragment of the large subunit of yRP-A (yRF-A) - 150, and for SEP1 + SF1 - two SEPI molecules per circular single-stranded DNA [ 30, 39].
an Mr 70 000 polypeptide. Based on amino acid sequencing data of the M r 34 000 polypeptide, we deduced that a significant portion of the NH2-terminus and probably some part of the COOH-terminus were missing probably due to unspecific proteolysis. Amino acid sequence homology revealed that the R P A I gene encoding this single-strand DNA binding protein is highly homologous to the large subunit of mammalian RP-A (RF-A). Replication Protein A (RP-A); also called Replication Factor A (RF-A) is a novel type of single-stranded DNA binding protein identified in higher eukaryotes as a factor essential for SV40 in vitro replication and is believed to be the equivalent of prokaryotic SSBs in eukaryotes [33-35]. RP-A (RF-A) is a three subunit protein with subunits of Mr 70 000, 32 000, and 14 000 showing high affinity for single-stranded DNA. The large Mr 70 000 subunit was demonstrated to be the DNA binding activity [36]. A very similar trimeric complex has been purified from the yeast S cerevisiae [37]. Again, the large s~abunit was identified as the DNA binding activity. Sequence comparisons established that the
S cerevisiae strand exchange protein 1 (SEPI) RPA1 gene encodes the large subt:~it of the S cerevisiae yRP-A (yRF-A) complex [32]. One DNA binding site of the Mr 70 000 subunit was therefore more precisely defined knowing the boundaries of the Mr 34 000 proteolytic fragment. Genetic analysis of insertion mutations in the S cerevisiae RPAI gene rc,vealed that it was essential for mitotic growth [32]. Cells lacking RPAI arrested as mononucleate, multiplied budded structures similar to cdc mutations [cdc4, 34; 38] believed to be defective in the initiation of chromosomal DNA synthesis. This phenotype is consistent with an involvement of yRP-A (yRF-A) in DNA replication analogous to the situation in higher eukaryotes.
Stimulatory Factor I (SF1) A second polypeptide has been identified and ourified in this approach. This activity has been termed SFI (Stimulatory Factor 1), as its presence vastly increased the formation of joint molecules in reactions with limiting amounts of SEP1 [39]. SF1 was purified using the in vitro stimulation assay described above and was found to co-purify with SEPI over two chromatographic steps, separating during anionexchange chromatography. As illustrated in figure 2, high and stoichiometric amounts of SFI (1 M, 33 000 monomer per 20 nucleotides of single-stranded DNA) lowered the requirement for SEP1 in the strand exchange reaction to about 1 SEP1 monomer per single-stranded substrate DNA molecule. This represents a roughly 300-fold stimulation as opposed to a 3-4 fold stimulation by the Mr 34 000 fragment of RPA1. This stimulation was specific to SEPI as E coli recA protein was not stimulated by SFI, contrary to the situation with the M~ 34 000 polypeptide. The reaction requirements of the SEPI-SFI catalyzed reaction remained unchanged, in particular the reaction still did not require a high energy cofactor. Electron microscopic analysis of the reaction IA~ c~r2 products of ~t,~c otzP1-SF1 catalyzed reaction demonstrated the occurrence of all three forms of joint molecules, namely cy- and o~-structures, as well as an open circular end product. Therefore, the reaction products of the stimulated and unstimulated reaction were indistinguishable in this respect. Strand exchange proteins such as SEP1, STPot, or recA are unspecifically stimulated by conditions that precipitate the substrate DNA, ie by the addition of spermidine or basic proteins such as histones [10, 12, 39-41]. While precipitation of the DNA was certainly one factor in the observed stimulation of SEPI by SFI, it cannot explain the available data. SFI stimulated SEP 1 at least 1 order of magnitude more than any other condition (addition of spermidine or histones).
273
In addition, the effect was specific for SEPI. To directly exclude that the SFI effect was entirely due to unspecific substrate precipitation, reaction conditions were achieved where the DNA was not precipitated but a significant stimulatory effect of SFI was observed [39]. SF1 protein was also characterized in the absence of SEPI [42]. The hydrodynamic properties of the polypeptide indicated that SF1 in solution is a monomer of asymmetric shape (axial ratio 8:1) with a native molecular weight of 31 000 Da. Filterbinding assays showed that SF1 is a DNA binding protein with a preference for single-stranded DNA. Fluorescence spectroscopic techniques determined the binding site size of SF1 to be 8 nucleotides of single-stranded DNA and a dissociation constant, KD. of 2.83 X 10-6 M. The binding to single-stranded DNA appeared to be ngn-co-operative. In addition, the SFI polypeptide catalyzed the simplest pairing reaction, the renaturation of two complementary single-stranded DNAs, in the absence ef SEP1. Several models can be developed to explain the mechanism of stimulation of SEPI by SF1. Generally, SF1 could act directly or indirectly in the strand exchange reaction. An example for stimulation by indirect action is the way prokaryotic SSBs stimulate recA which is the consequence of the disruption of secondary structures in the single-stranded DNA substrate mediated by the binding of the SSB to the nucleic acid [31]. The biochemical properties of SF1 and the specificity of stimulation are not compatible with this model. The capability of SFI to renature single-stranded DNA points to a more direct involvement of this protein in the strand exchange reaction. One model would suggest a role of SF1 in the initiation of synapsis and SEP1 promoting branch migration in a subsequent phase of the reaction. This view requires that SEP1 molecules recycle within one growing hybrid DNA or recycle to new substrates after completion of one joint, since only 1-2 SEP1 molecules are required for each joint molecule formed containing several kbp of hybrid DNA. Alternatively, SEPI might initiate synapsis with SF1 promoting branch migration. In this case, only a small number of SEP! molecules is required, while a much higher stoichiometry of SF1 is needed. This view most closely reflects the experimental data without invoking recycling of proteins within or between substrates which we imagine is a process that will require energy as in the case of recA [43]. A more detailed analysis of the interaction of SEPI and SFI must be awaited to develop more specific models. In addition, the recent cloning of the gene encoding SFI (unpublished result of DN and RDK) combined with reverse genetics might reveal a more precise role of SFI in hybrid DNA formation in vitro and in vivo.
274
WD Heyer et ai
Discussion
A number of proteins exerting strand exchange activity have been purified from the yeast S cerevisiae [SEPI: 9; STPo~: 10: DPA: 11: STPI3: 12] and the question becomes important as to whether they represent different proteins or not. The apparent molecular weight differences are not a reliable parameter for comparison, because of the severe proteolysis problems in yeast (ie see above for SEPI ). SEPI, as described above, has been purified from mitotic cells as an Mr 132 000 polypeptide [9]. Recently, Dykstra et al [ 12] described the purification of the same activity from mitotic cells as an Mr 180 000 form and renamed it STP[~. The available evidence shows that the two activities are the same protein with STP[~ (Mr of 180 000) being the full length form of SEPI. The two proteins are both recognized by monoclonal antibodies directed against SEPI. Furthermore, the two proteins show identical proteolytic degradation patterns. Above all, the genes encoding SEPI and STP[~ are the same based on sequence comparisons. Given the results of the immunological studies showing that the full length form of SEPI appears to be the authentic form in the cell, we conclude that the proteolysis leading to the Mr 132 000 form has been a purification artefact with no likely in vivo significance. However, the construction of clones expressing the truncated form of SEP1 and their functional analysis has to confirm this view. STPct has been purified from meiotic cells showing an apparent Mr of 38 000 [10]. In considering the properties of STP~a, it is important to note that ill,b,
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appear to have been demonstrated in the absence of ag~egating agents. Immunological studies by Dykstra et al [12] clearly distinguish STPct from SEP1 using polyclonal antisera against the two proteins. Furthermore, they demonstrated the presence of SEP 1 protein in S cerevisiae strain carrying a disruption of the gene D S T I gene supposedly encoding STPct. Based on protein sequence comparisons, the third strand exchange activity in S.cerevisiae, DPA [i 1], appears to be distinct from SEPI (unpublished result of RDK and K McEntee). Therefore, in the yeast S cerevisiae there is good evidence to suggest that at least three distinct proteins exert strand exchange activity. The existence of redundant activities complicates the interpretation of the phenotype of mutations in ary single gene encoding a strand exchange protein. If the function of the different proteins overlap, a mutational analysis of only one of the genes may not reveal the entire in vivo function of the protein. Therefore, the results of the preliminary analysis of insertion mutations in the SEP1 gene must be interpreted with this caveat.
All S cerevisiae strand exchange activities share the characteristic that they do not require a high energy co-factor to catalyze the reaction in vitro [9-11 ]. This property is shared among all purified strand exchange proteins from eukaryotes [23, 24] except for the U maydis recl protein [8]. Several activities were found to be dependent on the presence of ATP in crude extract systems or in partially purified fractions [ 15, 16, 19-21 ]. However, due to the presence of many proteins in these fractions it is difficult to assess whether the ATP dependance is an intrinsic property of the strand exchange protein or not. In fact, an activity from mammalian cells previously described as ATP dependent [21 ] turned out to be ATP independent upon final purification of the protein [24]. The observed ATP dependance of the reaction in earlier fractions might have been the result of a limiting stimulating activity that required a high energy cofactor. The absence of an energy requirement of many eukaryotic strand exchange activities was puzzling and in contrast to the concepts developed over the years with the E coli recA protein that strand exchange is an energy consuming reaction. Initially, it was suggested that recA protein had to bind and hydrolyze ATP in order to form hybrid DNA [44]. Experiments employing the non-hydrolyzable analog of ATP, ATP-7-S, demonstrated that recA could bind to DNA and form unstable (paranemic)joints [45]. Therefore, it was suggested that the energy requiring step in the reaction is the conversion from unstable (paranemic) joints to stable (plectonemic)joints. In addition, branch migration was suggested to require ATP hydrolysis [44]. In general, the absence of an energy requirement does not pose a paradox, since no catalytic turnover has been demonstrated for any of the ATP independent strand exchange proteins. In fact, the high and stoichiometric amounts of protein required for the reaction suggests a non-catalytic mode of action. Recently, Kowalczykowski and colleagues [43] made the surprising observation of extended hybrid DNA formation by the recA protein m the presence of ATP-~,-S which was hydrolyzed to a minimal extent during the reaction (maximally 0.003 ATP-'t,-S molecules hydrolyzed per base pair hybrid DNA formed). They suggested that ATP hydrolysis is only required for product release by the recA protein. This product release step could be achieved in vitro by the standard treatment of the reaction with SDS/EDTA prior to gel electrophoresis in the case of the ATP independent strand exchange proteins [43]. Thus, the apparent paradox between the eukaryotic ATP independent proteins and recA could be reconciled by suggesting a functional separation of DNA-strand-transfer (ATP not required) and catalytic-turnover (ATP required) activities into physically distinct polypeptides in eukaryotes [43].
S cerevisiae strand exchange protein I (SEPI)
Acknowledgments This work was supported by the National Institutes of Health Grants GM29383 to RDK WDH was supported by postdoctoral fellowships from the Swiss National Science Fo-'mdation (No 85BE02) and from the Helen Hay Whitney Foundation (No F557). AWJ was supported by a postdoctoral fellowship from the National Institutes of Health (IF32GMI3594-01). DNN was supported by a postdoctoral fellowship from the American Cancer Society (PF-3218). We thank Dr A Sugino for sharing results and reagents that allowed the identification of SEP1 and STP[3 as the same protein. We thank Dr K McEntee for the sharing results to distinguish SEP1 from DPA.
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14 15
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Symington LS, Fogarty LM, Kolodner R (1983) Genetic recombination of homologous plasmids catalyzed by cellfree extracts of Saccharomyces cerevisiae. Cell 35. 805-813 Heyer WD, Evans DH, Kolodner RD (1988) Renaturation ,,¢ r ~ A by a . . . . ,,u,,,mvt ~-.~ cerevi~iae protein that catalyzes homologous pairing and strand exchange. J Biol Chem 263, 15189-15195 Tsang SS, Chow SA, Radding CM (1985) Networks of DNA and RecA protein are intermediates in homologous pairing. Biochemistry 24, 3226-3232 Chow SA, Radding CM (1985) Ionic inhibition of formation of RecA nucleoprotein networks blocks homologous pairing. Proc Natl Acad Sci USA 82. 5646-5650 Pugh BF, Cox MM (1988) General mechanism for RecA protein binding to duplex DNA. J Mol Bio1203.479--493 Heyer WD, Kolodner RD (1989) Purification and characterization of a protein from Saccharomyces cerevisiae that binds tightly to single-stranded DNA and stimulates a cognate strand exchange protein. Biochemistry 28. 2856--2862 Egner C, Azhderian E, Tsang SS, Radding CM. Chase JW (1987) Effects of various single-stranded-DNA-binding proteins on reactions promoted by RecA protein. J Bact 169, 3422-3428 Heyer WD, Rao MRS, Erdile LF, Kelly TJ, Kolodner RK (1990) An essential Saccharomvces cerevisiae singlestranded DNA binding protein is laomologous to the large subunit of human RP-A. EMBO J 9, 2321-2329 Wobbe CR, Weissbach L, Borowiec JA, Dean FB. Murakami Y, Bullock P, Hurwitz J (1987) Replication of simian virus 40 origin-containing DNA in vitro with
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WD Heyer et al purified proteins. Proc Nat1 Acad Sci USA 84, 1834-! 838 Fainnan MP, Stiilman B (1988) Cellular factors required lbr multiple stages of SV40 DNA replication in vitro EMBO J 7, 1211-1218 Wold MS, Kelly TJ (1988) Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci lISA 85, 2523-2527 Wold MS, Weinberg DH, Virshup DM, Li JJ, Kelly TJ (1989) Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem 264, 2801-2809 Brill SJ, Stillman B (1989) Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nano'e 342, 92-95 Pringle JR, Hartwell LH (1981) The Saccharomyces cerevisiae cell cycle. In: The molecular biology of the yeast
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(Strathern JN, Jones EW, Broach JR, eds) CSH, Cold Spring Harbor Laboratory, 97-142 39 Norris DN, Kolodner RD (1990a) Purification of a strand exchange stimulatory factor from Saccharomw'es cerevisiae. Biochemiso T 29, 7903-7911
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Hamatake RK, Dykstra CC, Sugino A (1989) Presynapsis and synapsis of DNA promoted by the STP~ and singlestranded DNA-binding proteins from Saccharomyces cerevisiae. J Biol Chem 264, 13336-13342 Shibata T, DasGupta C, Cunningham RP, Radding CM (1980) Homologous pairing in genetic recombination: Formation of D-loops by combined action of recA protein and a helix destabilizing protein. Proc Nat Acad Sci USA 77, 2606-2610 Norris DN, Kolodner RD (1990b) Interaction of a Saccharomvces cerevisiae strand exchange stimulatory factor witti DNA. Biochemiso'y 29, 7911-7917 Menetski JP, Bear DG, Kowalczykowski SC (1990) Stable heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc Natl Acad Sci USA 87, 21-25 Cox MM, Lehman IR (1981) RecA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Nati Acad Sci USA 78, 3433-3437 Riddles PW, Lehman IR (1985) The formation of plectonemic joints by the recA protein of Escherichia coli. Requirement for ATP hydrolysis. J Biol Chem 260, 170-173
© SociEt6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris
269
Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro WD Heyer*, AW Johnson, DN Norris, D Tishkoff, RD Kolodner** Department of Biological Chemistry and Molectdar Pharmacology, Harvard Medical School," Division ~" Cell and Molecldar Biology, Dana-Father Cancer Institute. 44 Binne v Street, Boston. M4 02 i 15 USA
(Received 29 October 1990; accepted 25 January 1991 )
Summary - - RecA-like activities that can form hybrid DNA ill vitro have been identified in a wide variety of organisms. We have previously described the strand exchange protein 1 (SEPI) from the yeast Saccharomyces cerevisiae that can form hybrid DNA in vitro. Purified as an Mr 132 000 polypeptide, recent molecular and immunological studies have now shown that the native form is an Mr 175 000 polypeptide containing strand exchange activity. The gene encod;ng SEPI has been cloned and sequenced. The primary sequence failed to reveal any significant sequence homology to other sequences in data base searches, hi vivo SEPI was found to be essential for normal meiosis as cells containing a homozygous insertion mutation in the SEPI gene failed to sporulate. In order to identify additional factors that are involved in hybrid DNA formation in S cerevisiae, we used an in vitro stimulation assay to identify proteins that reconstitute strand exchange activity in reactions containing limiting amounts of SEPI. We have identified two proteins that functionally interact with SEPI. First, an Mr 34 000 single-stranded DNA binding protein stimulated the reaction by lowering the requirement for SEPI about 3--4 fold. This protein is a fragment of the large subunit of a hetero-trimeric complex called yRP-A (yRF-A) which is thought to be the functional eukaryotic equivalent of single-stranded DNA binding proteins in prokaryotes. The gene encoding this protein (RPAI) is essential for growth. Second, an Mr 33 000 polypeptide, termed Stimulatory Factor 1 (SF1), dramatically stimulated the SEPI catalyzed reaction by lowering the requirement for SEPI about 300 fold. This protein specifically stimulated SEPI in a way that is quantitatively and qualitatively different from previously found stimulation of strand exchange proteins. S cerevisiae / recombination / strand exchange / hybrid DNA
Introduction
Genetic recombination is a fundamental cellular process which is implicated in a) the generation of genetic diversity, b) the repair of DNA damage, c) the homologous alignment of chromosomes before meiotic cell division, and d) the generation of genomic alterations that lead to changes in gene expression. Based mainly on fungal tetrad data, molecular models have been developed to account for the observed consequences of recombination events [1-3]. C o m m o n to all these models is the central role of the hybrid DNA intermediate. In fungi, hybrid D N A can be directly traced in the phenomenon called PMS (Post Meiotic S_egregation) where a DNA mismatch originating from the hybrid DNA has escaped mismatch repair, resulting in an impure *Present address: Institute of General Microbiology, BaltzerStr.4, CH-3012 Bern, Switzerland **Correspondence and reprints
meiotic product that will segregate the characters upon the first mitosis usmg proper genetical techniques. Thus, PMS can be a direct measure of hybrid D N A formation giving firm evidence for this intermediate [4]. The identification of the recA gene in Escherichia colt [5] and the functional analysis of the recA gene product further established the pivotal importance of the hybrid D N A intermediate in homologous recombination [6, 7]. The mechanics of hybrid DNA formation in vitro by the recA protein have been studied in great detail. Often the model reaction shown in figure 1 has been used. The assay makes use of simple DNA substrates with easy detection of the reaction products by gel electrophoresis. The confirmation by electron microscopy of the structure of the joint molecules as shown in figure 1 is essential. The ease of the strand exchange assay has prompted the search for r e c A - l i k e activities that can form hybrid D N A in vitro in m a n y organisms. Such activities have been detected in a wide variety
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Fig 1. Hybrid DNA assay: the strand exchange reaction. Linear double-stranded DNA (MI3 replicative form) and circular single-stranded DNA (M13 viral DNA) are the substrates. The products of a strand exchange reaction are collectively called joint molecules. Electron microscopic analysis can easily distinguish between ~- and s-forms and the end product, open circular DNA. The region of hybrid
Using the assay illustrated in figure 1 we have previously purified the strand exchange protein, SEP 1, from mitotic S cerevisiae cells [9]. The Mr 132 000 form of SEPI formed hybrid DNA in the strand exchange reaction with the polarity shown in figure 1, ie 3' to 5', like E.coli recA protein. SEPI was requ;red in high and stoichiometric amounts in this reaction of 1 M r 132 000 monomer per 12 nucleotides of single-stranded DNA. The formation of joint molecules required the substrates to be homologous. The
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overhang or blunt, had no influence on the efficiency of the reaction. The reaction requirements were rather simple, essentially only the presence of the divalent cation Mg++ was required. In particular, the strand exchange was catalyzed in the absence of any requirement for a high energy cofactor such as ATP. This is in apparent contrast to the situation with recA, which was thought to require ATP hydrolysis to catalyze strand exchange [7]. In addition, SEP1 neither bound ATP, as indicated by photoaffinity-labelling experiments using 8-azido ATP, nor exhibited ATPase activity (WDH and RDK., unpublished results). The absence of an energy requirement does not pose an energetic paradox, as there was no indication for catalytic turnover of the protein. Electron microscopic analysis of the reaction products demonstrated the presence of all three classes of joint molecules as shown in figure 1, namely t~- and t~-structures as well as the endproduct, open circular double-stranded DNA. The physical demonstration of s-structures eliminated the possibility that the formation of joint molecules was generated by the action of a nuclease. The average length of hybrid DNA formed was measured to be 4.1 kbp using a 7.2 kbp substrate.
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of organisms including fungi [Ustilago maydis: 8; S cerevisiae: %12], insects [Drosophila melanogastel': 13-14], mammals [15-21], and 91ants [16, 22]. However, only the fungal activities (cited above), one protein from D melanogaster [23], and one protein from man [24] have been purified to homogeneity. The purified proteins not only allow detailed biochemical characterization of the activity, but also open an avenue to clone the corresponding genes. The gene/protein sequences will provide valuable information on the phylogenetic relationship of these activities and possibly clues about the structurefunction relationship of these proteins. The occurrence of strand exchange activities in this wide range oi" organisms suggests some universal mechanisms in the formation of hybrid DNA. We have taken the yeast S cerevisiae as a model system to study the mechanism(s) of homologous recombination using a biochemical approach [25].
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A number of additional activities are associated with SEP1 that are important for its action in the formation of hybrid DNA in vitro and possibly in vivo [26]. SEPI bound single-stranded DNA as well as double-stranded DNA, but bound double-stranded DNA with lower relative affinity. The binding of SEP1 to the single-stranded DNA rendered the nucleic acid resistant to digestion with nuclease S1 by forming a high molecular weight aggregate that effectively precipitated the DNA apparently excluding access of the nuclease. The formation of these high molecular weight aggregates appeared to be a kinetic intermediate in the formation of joint molecules with strand exchange taking place in these large aggregates. Furthermore, SEP1 catalyzed the renaturation of two complementary single-stranded DNAs, the most simple pairing reaction. The mechanism of the renaturation reaction seemed slightly different to that of the strand exchange reaction in that renaturation was not sensitive to the electrophilic blocking agent N-ethylmaleimide. These characteristics reveal many similarities to the recA protein. RecA binds to single-stranded DNA in a co-operative manner and also renatures complementary single-stranded DNA strands, which can occur even in an ATP independent fashion under certain reaction conditions [7]. In addition, recA causes DNA aggregation which was concluded to be an intermediate in DNA renaturation and strand exchange [27, 28]. Another difference is that SEP1 binds readily to double-stranded DNA, whereas recA has , , to Ua ,U.U, , kOt l ~.-.3.1. .. 1. . d i I U.a~.~ I~U . . . . a. . . k. .i n. .e .t i.c. . . harrier i n h ; n r i ; , ~ , E DNA at neutral pH [291. The gene encoding SEP1 has recently been cloned using degenerate oligonucleotides derived from protein sequencing data, and the DNA sequence has been established (DT and RDK, unpublished results). The sequence revealed an open reading frame with a coding potential for an Mr 175 000 polypeptide showing no significant primary sequence homology to any other protein in data base searches. The full length Mr 175 000 polypeptide has also been overproduced and purified. It was shown to exert strand exchange activity in the strand exchange assay (fig 1) with slightly higher specific activity than the Mr 132 000 form of SEP1 (AWJ and RDK, unpublished results). These data are in agreement with a recent study of Dykstra et al. [ 12] who have also identified the higher molecular weight form of SEP1. Several lines of evidence suggest that the Mr 175 000 polypeptide is the authentic form of SEPI. First, the open reading frame identifying the SEP1 gene has a coding potential for a polypeptide of this size. Second, NH2-terminal amino acid sequencing and proteolysis studies confirmed that in the M, 132 000 form of SEP1 the COOH-terminal - 40 000 kd were . . . . . . . . . . . .
missing. This part of the protein is predicted to be rather unorganized as it shows a high content of proline residues and was found to be protease sensitive. Third, monoclonal antibodies directed against the Mr 132 000 form of SEP1 detected the Mr 175 000 polypeptide in crude cell extracts. Monoclonal antibodies directed against SEP1 readily detected the Mr 175 000 polypeptide in crude cell extracts and identified SEPI as a rather abundant cellular protein. SEPI was found to be present throughout the mitotic cell cycle, during meiosis and was detected in spores. Indirect immunofluorescence light microscopy using anti-SEP1 monoclonal antibodies showed co-localization of SEP1 with the DNA specific fluorescent probe 4,6--diamidino-2-phenyiindole (DAPI) suggesting a nuclear localization of SEP1. (WDH, AWJ, and RDK, unpublished results) The preliminary analysis (DT and RDK, unpublished results) of insertion mutations in the SEP1 gene showed that the gene was not essential for mitotic growth. However the gene was important for mitotic growth as the cells carrying the mutation were clearly growing slower than wild-type. For meiosis, however, SEP1 was essential, since sepl mutants were defective in sporulation resulting in 0-3% ascus formation with 60% spore viability. During meiosis sepl mutant cells underwent an apparently normal DNA synthesis, but were blocked before they proceeded through meiosis I. Cells blocked at this stage could be returned to mitotic -rowth by n l n t i n a t h e m ran , a n n r r a n r ; ~ t a m a A ; . . . . ;*h . . . . significant loss of viability. Preliminary examination of the recombination phenotype of sepl mutant strains showed a very slight reduction (~ 5-fold) in prototroph formation from two his4 hetero-alleles during mitotic growth compared to wild-type. Induction of recombination during meiosis by a factor of 100 to 1000 is a hallmark of yeast genetics. Induction of meiotic prototroph formation from his4 hetero-alleles appeared normal in kinetics but were induced to a greater extent in sepl mutant strains. When mutant cells were examined for the level of prototroph formation after being left at the meiotic block for 24 h after induction of meiosis, they exhibited a 6--10-fold higher meiotic induction than wildtype cells. By this time wild-type cells had already have completed meiosis (ie sporulated) for about 10 h. These preliminary data suggest that the SEPI protein is essential for a successful meiosis, and that the meiotic block in sepl mutants occurs at a stage in meiosis where recombination can take place. An alternative explanation is that sepl mutations block a type of recombination required for meiosis, and that the recombination observed in sepl mutants is promoted by a parallel pathway. --
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Additional factors involved in hybrid DNA formation in vitro A long term biochemical goal in studying genetic recombination is the reconstitution of large parts or of the entire recombination reaction. We have chosen to start with the strand exchange reaction catalyzed by SEPI (fig I). The formation of hybrid DNA is central to genetic recombination, and we envisaged that multiple proteins may function in this reaction in :'ivo. In order to identify additional proteins that r,.re potentially involved in hybrid DNA formation, we have purified proteins that stimulate the SEPI catalyzed reaction in vio'o under conditions where the anaount of SEPI was limiting. The results of this in vio'o stimulation approach are schematically illustrated in figure 2. In practice, strand exchange reactions were set up with amounts of SEPI too low to sustain activity. Column tractions were systematically &ssayed l~or the presence of activities that reconstituted activity. Two proteins were identified and purified by this approach. Mr 34 000 single-stranded DNA binding protein [fragment of yRP-A (yRF-A)] The first protein was identified as an Mr 34 000 polypeptide that showed nucleic binding properties typical for single-stranded DNA binding proteins [30]. The polypeptide had high affinity for single-stranded DNA. Competition experiments showed no or much lower affinity for double-stranded DNA or RNA. The protein. DNA complexes between the Mr 34 000 polypeptide and single-stranded DNA were highly salt stable (elution point from single-stranded DNA cellulose - 1.2 M NaCI). The in vitro functional interaction of the Mr 34 000 polypeptide with SEPI resulted in stimulation of the SEPI catalyzed strand exchange reaction at an optimal stoichiometry of 1 monomer per 18 nucleotides of single-stranded DNA. As shown schematically in figure 2, the amount of SEP1 required to form joint molecules was lowered by 3-4-fold. E coli SSB could not elicit stimulation of SEP1. Conversely, the Mr 34 000 polypeptide stimulated recA as well as E coli SSB did. This is consistent with previous observations that recA can be stimulated by various heterologous singlestranded DNA binding proteins [31]. The Mr 34 000 alone was inert in the strand exchange reaction, only showing an effect when the amount of SEPI was limiting. In this case, both extent and rate of the SEP1 catalyzed reaction were stimulated. The gene encoding the Mr 34 000 polypeptide (RPA',~ was cloned using degenerate oligonucleotide probes and analyzed [32]. The open reading frame identifying the gene showed a coding potential for
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an Mr 70 000 polypeptide. Based on amino acid sequencing data of the M r 34 000 polypeptide, we deduced that a significant portion of the NH2-terminus and probably some part of the COOH-terminus were missing probably due to unspecific proteolysis. Amino acid sequence homology revealed that the R P A I gene encoding this single-strand DNA binding protein is highly homologous to the large subunit of mammalian RP-A (RF-A). Replication Protein A (RP-A); also called Replication Factor A (RF-A) is a novel type of single-stranded DNA binding protein identified in higher eukaryotes as a factor essential for SV40 in vitro replication and is believed to be the equivalent of prokaryotic SSBs in eukaryotes [33-35]. RP-A (RF-A) is a three subunit protein with subunits of Mr 70 000, 32 000, and 14 000 showing high affinity for single-stranded DNA. The large Mr 70 000 subunit was demonstrated to be the DNA binding activity [36]. A very similar trimeric complex has been purified from the yeast S cerevisiae [37]. Again, the large s~abunit was identified as the DNA binding activity. Sequence comparisons established that the
S cerevisiae strand exchange protein 1 (SEPI) RPA1 gene encodes the large subt:~it of the S cerevisiae yRP-A (yRF-A) complex [32]. One DNA binding site of the Mr 70 000 subunit was therefore more precisely defined knowing the boundaries of the Mr 34 000 proteolytic fragment. Genetic analysis of insertion mutations in the S cerevisiae RPAI gene rc,vealed that it was essential for mitotic growth [32]. Cells lacking RPAI arrested as mononucleate, multiplied budded structures similar to cdc mutations [cdc4, 34; 38] believed to be defective in the initiation of chromosomal DNA synthesis. This phenotype is consistent with an involvement of yRP-A (yRF-A) in DNA replication analogous to the situation in higher eukaryotes.
Stimulatory Factor I (SF1) A second polypeptide has been identified and ourified in this approach. This activity has been termed SFI (Stimulatory Factor 1), as its presence vastly increased the formation of joint molecules in reactions with limiting amounts of SEP1 [39]. SF1 was purified using the in vitro stimulation assay described above and was found to co-purify with SEPI over two chromatographic steps, separating during anionexchange chromatography. As illustrated in figure 2, high and stoichiometric amounts of SFI (1 M, 33 000 monomer per 20 nucleotides of single-stranded DNA) lowered the requirement for SEP1 in the strand exchange reaction to about 1 SEP1 monomer per single-stranded substrate DNA molecule. This represents a roughly 300-fold stimulation as opposed to a 3-4 fold stimulation by the Mr 34 000 fragment of RPA1. This stimulation was specific to SEPI as E coli recA protein was not stimulated by SFI, contrary to the situation with the M~ 34 000 polypeptide. The reaction requirements of the SEPI-SFI catalyzed reaction remained unchanged, in particular the reaction still did not require a high energy cofactor. Electron microscopic analysis of the reaction IA~ c~r2 products of ~t,~c otzP1-SF1 catalyzed reaction demonstrated the occurrence of all three forms of joint molecules, namely cy- and o~-structures, as well as an open circular end product. Therefore, the reaction products of the stimulated and unstimulated reaction were indistinguishable in this respect. Strand exchange proteins such as SEP1, STPot, or recA are unspecifically stimulated by conditions that precipitate the substrate DNA, ie by the addition of spermidine or basic proteins such as histones [10, 12, 39-41]. While precipitation of the DNA was certainly one factor in the observed stimulation of SEPI by SFI, it cannot explain the available data. SFI stimulated SEP 1 at least 1 order of magnitude more than any other condition (addition of spermidine or histones).
273
In addition, the effect was specific for SEPI. To directly exclude that the SFI effect was entirely due to unspecific substrate precipitation, reaction conditions were achieved where the DNA was not precipitated but a significant stimulatory effect of SFI was observed [39]. SF1 protein was also characterized in the absence of SEPI [42]. The hydrodynamic properties of the polypeptide indicated that SF1 in solution is a monomer of asymmetric shape (axial ratio 8:1) with a native molecular weight of 31 000 Da. Filterbinding assays showed that SF1 is a DNA binding protein with a preference for single-stranded DNA. Fluorescence spectroscopic techniques determined the binding site size of SF1 to be 8 nucleotides of single-stranded DNA and a dissociation constant, KD. of 2.83 X 10-6 M. The binding to single-stranded DNA appeared to be ngn-co-operative. In addition, the SFI polypeptide catalyzed the simplest pairing reaction, the renaturation of two complementary single-stranded DNAs, in the absence ef SEP1. Several models can be developed to explain the mechanism of stimulation of SEPI by SF1. Generally, SF1 could act directly or indirectly in the strand exchange reaction. An example for stimulation by indirect action is the way prokaryotic SSBs stimulate recA which is the consequence of the disruption of secondary structures in the single-stranded DNA substrate mediated by the binding of the SSB to the nucleic acid [31]. The biochemical properties of SF1 and the specificity of stimulation are not compatible with this model. The capability of SFI to renature single-stranded DNA points to a more direct involvement of this protein in the strand exchange reaction. One model would suggest a role of SF1 in the initiation of synapsis and SEP1 promoting branch migration in a subsequent phase of the reaction. This view requires that SEP1 molecules recycle within one growing hybrid DNA or recycle to new substrates after completion of one joint, since only 1-2 SEP1 molecules are required for each joint molecule formed containing several kbp of hybrid DNA. Alternatively, SEPI might initiate synapsis with SF1 promoting branch migration. In this case, only a small number of SEP! molecules is required, while a much higher stoichiometry of SF1 is needed. This view most closely reflects the experimental data without invoking recycling of proteins within or between substrates which we imagine is a process that will require energy as in the case of recA [43]. A more detailed analysis of the interaction of SEPI and SFI must be awaited to develop more specific models. In addition, the recent cloning of the gene encoding SFI (unpublished result of DN and RDK) combined with reverse genetics might reveal a more precise role of SFI in hybrid DNA formation in vitro and in vivo.
274
WD Heyer et ai
Discussion
A number of proteins exerting strand exchange activity have been purified from the yeast S cerevisiae [SEPI: 9; STPo~: 10: DPA: 11: STPI3: 12] and the question becomes important as to whether they represent different proteins or not. The apparent molecular weight differences are not a reliable parameter for comparison, because of the severe proteolysis problems in yeast (ie see above for SEPI ). SEPI, as described above, has been purified from mitotic cells as an Mr 132 000 polypeptide [9]. Recently, Dykstra et al [ 12] described the purification of the same activity from mitotic cells as an Mr 180 000 form and renamed it STP[~. The available evidence shows that the two activities are the same protein with STP[~ (Mr of 180 000) being the full length form of SEPI. The two proteins are both recognized by monoclonal antibodies directed against SEPI. Furthermore, the two proteins show identical proteolytic degradation patterns. Above all, the genes encoding SEPI and STP[~ are the same based on sequence comparisons. Given the results of the immunological studies showing that the full length form of SEPI appears to be the authentic form in the cell, we conclude that the proteolysis leading to the Mr 132 000 form has been a purification artefact with no likely in vivo significance. However, the construction of clones expressing the truncated form of SEP1 and their functional analysis has to confirm this view. STPct has been purified from meiotic cells showing an apparent Mr of 38 000 [10]. In considering the properties of STP~a, it is important to note that ill,b,
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appear to have been demonstrated in the absence of ag~egating agents. Immunological studies by Dykstra et al [12] clearly distinguish STPct from SEP1 using polyclonal antisera against the two proteins. Furthermore, they demonstrated the presence of SEP 1 protein in S cerevisiae strain carrying a disruption of the gene D S T I gene supposedly encoding STPct. Based on protein sequence comparisons, the third strand exchange activity in S.cerevisiae, DPA [i 1], appears to be distinct from SEPI (unpublished result of RDK and K McEntee). Therefore, in the yeast S cerevisiae there is good evidence to suggest that at least three distinct proteins exert strand exchange activity. The existence of redundant activities complicates the interpretation of the phenotype of mutations in ary single gene encoding a strand exchange protein. If the function of the different proteins overlap, a mutational analysis of only one of the genes may not reveal the entire in vivo function of the protein. Therefore, the results of the preliminary analysis of insertion mutations in the SEP1 gene must be interpreted with this caveat.
All S cerevisiae strand exchange activities share the characteristic that they do not require a high energy co-factor to catalyze the reaction in vitro [9-11 ]. This property is shared among all purified strand exchange proteins from eukaryotes [23, 24] except for the U maydis recl protein [8]. Several activities were found to be dependent on the presence of ATP in crude extract systems or in partially purified fractions [ 15, 16, 19-21 ]. However, due to the presence of many proteins in these fractions it is difficult to assess whether the ATP dependance is an intrinsic property of the strand exchange protein or not. In fact, an activity from mammalian cells previously described as ATP dependent [21 ] turned out to be ATP independent upon final purification of the protein [24]. The observed ATP dependance of the reaction in earlier fractions might have been the result of a limiting stimulating activity that required a high energy cofactor. The absence of an energy requirement of many eukaryotic strand exchange activities was puzzling and in contrast to the concepts developed over the years with the E coli recA protein that strand exchange is an energy consuming reaction. Initially, it was suggested that recA protein had to bind and hydrolyze ATP in order to form hybrid DNA [44]. Experiments employing the non-hydrolyzable analog of ATP, ATP-7-S, demonstrated that recA could bind to DNA and form unstable (paranemic)joints [45]. Therefore, it was suggested that the energy requiring step in the reaction is the conversion from unstable (paranemic) joints to stable (plectonemic)joints. In addition, branch migration was suggested to require ATP hydrolysis [44]. In general, the absence of an energy requirement does not pose a paradox, since no catalytic turnover has been demonstrated for any of the ATP independent strand exchange proteins. In fact, the high and stoichiometric amounts of protein required for the reaction suggests a non-catalytic mode of action. Recently, Kowalczykowski and colleagues [43] made the surprising observation of extended hybrid DNA formation by the recA protein m the presence of ATP-~,-S which was hydrolyzed to a minimal extent during the reaction (maximally 0.003 ATP-'t,-S molecules hydrolyzed per base pair hybrid DNA formed). They suggested that ATP hydrolysis is only required for product release by the recA protein. This product release step could be achieved in vitro by the standard treatment of the reaction with SDS/EDTA prior to gel electrophoresis in the case of the ATP independent strand exchange proteins [43]. Thus, the apparent paradox between the eukaryotic ATP independent proteins and recA could be reconciled by suggesting a functional separation of DNA-strand-transfer (ATP not required) and catalytic-turnover (ATP required) activities into physically distinct polypeptides in eukaryotes [43].
S cerevisiae strand exchange protein I (SEPI)
Acknowledgments This work was supported by the National Institutes of Health Grants GM29383 to RDK WDH was supported by postdoctoral fellowships from the Swiss National Science Fo-'mdation (No 85BE02) and from the Helen Hay Whitney Foundation (No F557). AWJ was supported by a postdoctoral fellowship from the National Institutes of Health (IF32GMI3594-01). DNN was supported by a postdoctoral fellowship from the American Cancer Society (PF-3218). We thank Dr A Sugino for sharing results and reagents that allowed the identification of SEP1 and STP[3 as the same protein. We thank Dr K McEntee for the sharing results to distinguish SEP1 from DPA.
References l 2 3 4 5 6 7 8 9
10
11
12
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