Two modes of FEN1 binding to PCNA regulated by DNA

The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 was still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreased both modes of interaction with PCNA and resulted in replication and repair defects in vivo.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta). Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.     

Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally,suggesting a role for polymerase epsilon in sister chromatid cohesion

The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.     

Lagging strand DNA synthesis by calf thymus DNA polymerases alpha, beta, delta and epsilon in the presence of auxiliary proteins.

By using a defined gapped DNA substrate that mimics a lagging strand of 230 nucleotides and that contains a defined pause site, we have analyzed calf thymus DNA polymerases (pol) alpha, beta, delta, and epsilon in the presence of the three auxiliary proteins proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) for their ability to complete an Okazaki fragment. Pol alpha alone could fill the gap to near completion, but was strongly stopped by the pause site. Addition of low amounts of RP-A resulted in an increased synthesis by pol alpha past the pause site. In contrast, high amounts of RP-A strongly inhibited gap filling by pol alpha. Further inhibition was evident when the two other auxiliary proteins, PCNA and RF-C, were added in addition to RP-A. Pol beta could completely fill the gap without specific pausing and also was strongly inhibited by RP-A. PCNA and RF-C had no detectable effect on pol beta. Pol delta, relied as expected, on all three auxiliary proteins for complete gap filling synthesis and could, upon longer incubation, perform a limited amount of strand displacement synthesis. Pol epsilon core enzyme was able to fill the gap completely, but like pol alpha, essentially stopped at the pause site. This pausing could only be overcome upon addition of PCNA, RF-C and E. coli single-stranded DNA binding protein. Thus pol epsilon holoenzyme preferentially synthesized to the end of the gap without pausing. Ligation of the DNA products indicated that pol beta core enzyme, pol delta and pol epsilon holoenzymes (but not pol alpha and pol epsilon core enzyme) synthesized products that were easily ligatable. Our results indicate that pol epsilon holoenzyme fills a defined lagging strand gapped template to exact completion and is able to pass a pause site. The data favour the hypothesis of Burgers (Burgers, P.M.J. (1991) J. Biol. Chem. 266, 22698-22706) that pol epsilon might be a candidate for the second replication enzyme at the lagging strand of the replication fork.     

The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis

Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.     

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon ) complex. Demonstration of a dimeric pol epsilon

Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication. A major form of pol epsilon purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol epsilon may be dimeric in vivo. Gel filtration of the Pol2p.Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.     

Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzyme. Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins would help identify region(s) involved in the assembly of the pol delta holoenzyme. Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared by swapping corresponding regions between the two proteins. In solution, all PCNA assembled into trimers, albeit to different extents. These PCNA variants were tested for stimulation of pol delta and in vitro replication of M13 and SV40 DNA as well as to stimulate the ATPase activity of replication factor C (RF-C). Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is required for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, indicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.     

Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31

Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.     

The DNA polymerase domain of pol(epsilon) is required for rapid,efficient, and highly accurate chromosomal DNA replication,telomere length maintenance,and normal cell senescence in Saccharomyces cerevisiae

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.     

Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.     

The extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication.

The herpes simplex virus DNA polymerase is composed of two subunits, a large catalytic subunit (Pol) and a smaller subunit (UL42) that increases the processivity of the holoenzyme. The interaction between the two polypeptides is of interest both for the mechanism by which it enables the enzyme to synthesize long stretches of DNA processively and as a possible target for the rational design of novel antiviral drugs. Here, we demonstrate through a combination of insertion and deletion mutagenesis that the carboxy-terminal 35 amino acids of Pol are crucial for binding UL42. The functional importance of the interaction was confirmed by the finding that a pol mutant defective for UL42 binding retained polymerase activity, but did not synthesize longer DNA products in the presence of UL42. Moreover, several association-incompetent mutants failed to complement the replication of a pol null mutant in a transient transfection assay, confirming that the Pol-UL42 interaction is necessary for virus replication in vivo and therefore a valid target for directed drug design.     

Proliferating cell nuclear antigen-dependent coordination of the biological functions of human DNA polymerase iota

Y-family DNA polymerases are believed to facilitate the replicative bypass of damaged DNA in a process commonly referred to as translesion synthesis. With the exception of DNA polymerase eta (poleta), which is defective in humans with the Xeroderma pigmentosum variant (XP-V) phenotype, little is known about the cellular function(s) of the remaining human Y-family DNA polymerases. We report here that an interaction between human DNA polymerase iota (poliota) and the proliferating cell nuclear antigen (PCNA) stimulates the processivity of poliota in a template-dependent manner in vitro. Mutations in one of the putative PCNA-binding motifs (PIP box) of poliota or the interdomain connector loop of PCNA diminish the binding between poliota and PCNA and concomitantly reduce PCNA-dependent stimulation of poliota activity. Furthermore, although retaining its capacity to interact with poleta in vivo, the poliota-PIP box mutant fails to accumulate in replication foci. Thus, PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and translesion DNA synthesis polymerases to ensure genome integrity.     

In vivo reconstitution of Saccharomyces cerevisiae DNA polymerase epsilon in insect cells. Purification and characterization.

DNA polymerase epsilon (pol epsilon) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol epsilon has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol epsilon complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol epsilon complex and truncated Pol2p (140 kDa), as was observed initially with the pol epsilon purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol epsilon is functionally identical to native pol epsilon purified from yeast and is therefore useful for biochemical characterization of the interactions of pol epsilon with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Pol2p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.     

A single domain in human DNA polymerase iota mediates interaction with PCNA: implications for translesion DNA synthesis

DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, eta, iota, kappa, and Rev1, of which Pols eta, iota, and kappa have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Poldelta, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent K(m) for the nucleotide. Here we show that Poliota interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Poliota is quite unlike that in Poldelta, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site.     

The Pol30-K196 residue plays a critical role in budding yeast DNA postreplication repair through interaction with Rad18

PCNA plays critical roles in DNA replication and various DNA repair pathways including DNA damage tolerance (DDT). In budding yeast Saccharomyces cerevisiae, DDT (aka DNA postreplication repair, PRR) is achieved by sequential ubiquitination of PCNA encoded by POL30. Our previous studies revealed that two Arabidopsis PCNA genes were able to complement the essential function of POL30 in budding yeast, but failed to rescue the PRR activity. Here we hypothesize that a certain amino acid variation(s) is responsible for the difference, and identified K196 as a critical residue for the PRR activity. It was found that the pol30-K196V mutation abolishes Rad18 interaction and PRR activity, whereas nearby amino acid substitutions can partially restore Rad18 interaction and PRR activity. Together with the Pol30-Ub fusion data, we believe that we have identified a putative Rad18-binding pocket in Pol30 that is required for PCNA monoubiquitination and PRR.     

Dominant mutations in three different subunits of replication factor C suppress replication defects in yeast PCNA mutants

To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs(-)) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21(WAF1/CIP1) peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs(-) mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol delta-dependent DNA synthesis, which is consistent with an increase in processivity.     

Human DNA polymerase epsilon colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.

DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol epsilon foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the G(1) phase of cycling cells. It is hypothesized from these observations that pol epsilon and PCNA have separate but associated functions early in S phase and that pol epsilon participates with PCNA in DNA replication late in S phase.     

Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.