INTRODUCTION

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Transformation of fungi with plasmid DNA has yielded important insights into the mechanisms of recombination and double-strand break repair (DSBR). Early studies by Hinnen et al. (17) provided evidence for integration of circular nonreplicating plasmids by homologous recombination into the yeast genome. In a subsequent study, Orr-Weaver et al. (38) elaborated more thoroughly the way in which circular and linear nonreplicating DNA molecules recombine with homologous chromosomal sequences. They showed that DNA ends are highly recombinogenic and interact directly with homologous sequences. If two restriction cuts are made within a plasmid region homologous to chromosomal DNA, thereby producing a double-strand gap, the resulting deleted linear plasmids transform at a high frequency and are faithfully repaired during the integration process. Using linear replicating plasmids, Orr-Weaver and Szostak (37) reported the recovery of approximately equal numbers of integrated and nonintegrated plasmids and concluded that gene conversion by double-strand gap repair can occur either with or without crossing over. These studies formed the basis for the DSBR model (65). They also observed circularization of linear plasmid DNA, suggesting the presence of additional, recombination-independent repair pathways. Subsequent studies of plasmid gap repair in Saccharomyces cerevisiae and Ustilago maydis indicated a lower association of crossing over with gene conversion (12, 44). Studies in Drosophila and mouse cells have also shown a very low association of crossing over (<5%) during DSBR (35, 48).

When plasmids capable of autonomous replication are cut within regions that have no homology to yeast genomic sequences, repair of the break can occur by nonhomologous end joining (7). In yeast, the efficiency of this process is dependent on the types of ends produced. Cohesive ends are efficiently repaired by precise end joining in a reaction dependent on HDF1, HDF2, MRE11, RAD50, XRS2, DNL4, and LIF1 (31, 32, 51, 69). Cohesive ends generated in genomic DNA by either HO or EcoRI endonucleases are also efficiently repaired by the end-joining pathway, indicating that plasmid and chromosomal breaks are repaired by similar mechanisms (23). Repair of blunts ends is inefficient in wild-type cells (7). This contrasts with mammalian cells, which show efficient joining of a variety of DNA ends (50).

DNA repair-deficient strains have proven useful for understanding the genetic control of end joining, and the ease of recovering plasmids has allowed a molecular analysis of the type of end-joining event. Although the effects of mutations in DNA repair genes on mating-type switching (28, 60), direct repeat (25, 30), and inverted-repeat recombination (1, 45, 46) have been studied extensively, very few studies have dealt with the role of RAD genes in homology-dependent plasmid double-strand gap repair. The initial studies of plasmid gap repair demonstrated an essential role for RAD52 (38) and subsequent studies showed a requirement for RAD50, RAD53, RAD54, and RAD57, but the repair events were not analyzed in detail (14, 15, 42).

Although all of the genes of the RAD52 epistasis group are required for the repair of ionizing radiation-induced DNA damage, the mutants show considerable heterogeneity in recombination assays. RAD52 has a unique position within the group in that it is required for most spontaneous and induced mitotic recombination events, for the formation of joint molecules in the rDNA locus, and for single-strand annealing (46, 59, 73). The rad51, rad54, rad55, and rad57 mutants form a subgroup with similar phenotypes. In these mutants, joint molecules are still detected at the rDNA locus, and they are proficient at several double-strand break (DSB)-initiated and spontaneous mitotic recombination events (45, 60, 73). For example, although natural mating-type switching is lethal in rad51 mutants, repair of an HO endonuclease-induced DSB can occur under certain circumstances if the donor sequence is unsilenced and on a plasmid (60). Furthermore, a DSB introduced into one allele of the MAT locus in rad51 diploids can be efficiently repaired by strand invasion and replication primed from the invading strand to restore the chromosome arm (27). Single-strand annealing is also independent of RAD51, RAD54, RAD55, and RAD57 (19).

Rad51 has significant homology to bacterial RecA proteins and catalyzes DNA strand exchange in vitro (54, 64). Rad54 and the Rad55-Rad57 heterodimer enhance the efficiency of the Rad51-mediated strand exchange reaction, consistent with genetic studies indicating similar phenotypes of the respective mutants (20, 43, 45, 63). Rad52 stimulates the Rad51-promoted strand exchange reaction by overcoming the inhibitory effects of replication protein A (6, 36, 55, 62). This is consistent with studies showing physical interactions between these proteins (40, 54) and genetic analysis indicating that RAD52 is epistatic to RAD51 (46). The observation that high levels of certain types of recombinational repair can occur in the absence of RAD51 suggests that alternate mechanisms for homologous pairing and strand invasion exist in S. cerevisiae. RAD59 was identified by its requirement for RAD51-independent mitotic recombination of inverted repeats (4). However, Rad59 shows 28% identity to Rad52 instead of homology to the RecA family of proteins. Although RecA-like proteins have formed the paradigm for homologous pairing and strand exchange, recent studies suggest that a different class of proteins, exemplified by bacteriophage lambda  protein, provide an alternate pathway for recombinational repair (71, 72). Rad52 shows no primary sequence homology to  protein, but both proteins form ring structures and catalyze strand annealing in vitro (22, 34, 41, 56).

Two alternative hypotheses for RAD51-independent recombination have been suggested. First that RAD51, RAD54, RAD55, and RAD57 gene products do not play a direct role in recombination but instead are required to facilitate DNA strand invasion into otherwise inaccessible sequences (60). This hypothesis was put forward to explain the occurrence of RAD51-independent DSBR when the donor for repair was expressed and plasmid borne. If the hypothesis presented by Sugawara et al. (60), is correct, we would expect rad51 mutants to be defective in all assays that involve repair from a chromosomal donor but not when the donor sequences are expressed and on a plasmid. Second, an alternative explanation to the donor accessibility model is that recombination is RAD51 independent when the event can be resolved as a crossover. Based on evidence obtained from inverted-repeat recombination experiments (45, 46), we proposed that the RAD51 pathway, including RAD54, RAD55 and RAD57, leads primarily to noncrossover recombinants and is not involved in the recombination pathway that results in crossovers. A system for intermolecular plasmid gap repair was developed to test these hypotheses.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. The source of the gene encoding O-acetylhomoserine-O-acetylserine sulfhydralase, MET17 (21), was plasmid pGC3 (ATTC 87440), which contains a 2.5-kb SpeI fragment of the genomic MET17 locus cloned into pRS414 (57). To obtain a restriction fragment length polymorphism nonsense mutation marker (met17-s), oligonucleotide-directed insertion mutagenesis was performed on the unique SnaBI site in MET17 of plasmid pSB99 (see Fig. 1, nucleic acid position 943) using the Quik Change site-directed mutagenesis kit (Stratagene). Plasmid pSB99 was constructed by cloning the 1.8-kb XbaI MET17 fragment from pGC3 into the XbaI site of pBluescript (Stratagene). Plasmid pSB99 was used as the template and oligonucleotides oSB2 (5'-AGAACAATCCCCATAACGTATCTTGGGTTTC-3') and oSB1 (5'-AAACCCAAGATACGTTATGGGGATTGTTC-3') were used to direct the sequence change. The nucleotides in the oligomers that caused the PCR-mediated mutation of the SnaBI site (TACGTA) are underlined. Plasmids derived from the mutagenesis were screened for the absence of a SnaBI site and candidates were further analyzed by DNA sequencing to confirm that a stop codon was introduced (see Fig. 1). The mutagenized plasmid pSB99 was named pSB99-1. Plasmid pSB112 was constructed by replacing the XbaI-XbaI MET17 fragment in pGC3 with the mutagenized MET17 of pSB99-1. To create plasmid pSB115, used for the two-step replacement of the chromosomal MET17 locus by met17-s, a BamHI-NotI met17-s fragment was isolated from pSB112 and ligated to the BamHI-NotI fragment of the integrating vector pRS406 (57).

r_K m_K⁺

Media and strains. All media were prepared as described previously (53) with minor modifications. Synthetic complete (SC) medium lacked cysteine, since this amino acid can be converted into methionine via MET17-independent pathways (10). Selective media lacking 1 amino acid (aa) are designated SCaa, e.g., selection for LEU2 disrupted genes was performed in SCLeu which is SC with all the amino acids W303 needs for growth except leucine. Lead (Pb²⁺) plates were prepared by dissolving 3 g of peptone, 5 g of yeast extract, 200 mg of ammonium sulfate, and 40 g of glucose and suspending 20 g of agar in 1 liter of water. The suspension was autoclaved, the agar solution was cooled to 55°C, 2 ml of lead nitrate, Pb(NO₃)₂ (0.5 mg/ml of water), was added, and the solution was mixed vigorously before the plates were poured (10). Standard genetic techniques were used to manipulate yeast strains (53).

Plasmid DNA gap repair assays. Plasmids to be used as substrates in the gap repair assay were digested with BspEI and EcoNI (Fig. 1), and the linear DNA was gel purified. Transformation was performed by the lithium acetate transformation method (53) with 100 ng of gapped plasmid (gap repair substrate) or 100 ng of uncut plasmid (transformation efficiency control) in the presence of 50 µg of denatured salmon sperm DNA as carrier DNA. The transformed cells were diluted and plated onto SCUraMet and SCUra media. The colonies on both plates were counted after incubation at 30°C for 3 days. The amounts of DNA used were determined to be in the linear range for uptake of DNA. The specific gap repair frequency was calculated as the number of Met⁺ Ura⁺ recombinants per microgram of transformed linearized DNA divided by the total number of Ura⁺ Met⁺ transformants per microgram of appropriate circular transformation control DNA. As a control for undigested plasmid DNA contamination of the gapped substrate, a second yeast strain containing a complete deletion of the MET17 ORF was used as a host for transformation. The rare Met⁺ transformants arising from this strain were considered to be due to contamination of the gapped DNA with uncut plasmid, and this number was deducted from the number of Met⁺ transformants obtained in the experimental strain. The gap repair experiments were repeated at least twice for each substrate and strain, and the mean gap repair frequencies are presented.

Molecular analysis of gap repair recombinants. The mode of recombination during gap repair was determined by Southern blot analysis. Total DNA was extracted from 5-ml cultures of individual transformants (53) and digested with BamHI-SnaBI, and fragments were separated by electrophoresis through 0.7% agarose. DNA was transferred to a nylon membrane (Hybond-N; Amersham) and hybridized with a 1.5-kb BamHI-SnaBI MET17 fragment isolated from plasmid pGC3 that was ³²P labeled for use as the probe.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Rationale for the gap repair system. A set of recombination reporter substrates was constructed by subcloning the MET17 gene into plasmids of the pRS400 series containing the URA3 marker (57). These plasmids were gapped within the MET17 gene and used as recipients for gap repair during transformation into host strains. The gap was made in the plasmid by deleting a 238-bp fragment from the MET17 ORF with the restriction enzymes BspEI and EcoNI (Fig. 1). The restriction enzymes produce overlapping but noncomplementary ends that should be poor substrates for end-joining ligation reactions. The donor sequences for DNA repair contained a nonsense mutation at the SnaBI site of the chromosomal MET17 locus (met17-s). Since the met17-s mutation lies downstream of the region that covers the gap in the plasmid, Met⁺ transformants can arise by repair of the gap using chromosomal information (Fig. 2). Plasmid-by-plasmid gap repair was studied with the same rationale except that the met17-s donor sequence was located on a circular centromeric TRP1 plasmid that was introduced into a host strain in which the complete chromosomal MET17 ORF was replaced by the ADE2 ORF. Selection for the TRP1 plasmid was maintained throughout the plasmid by plasmid gap repair assay. The use of both chromosomal and plasmid donors was to test the hypothesis that RAD51 and RAD57 are not required if the donor sequence for gap repair is expressed and plasmid borne.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Physical map of MET17, the molecular structure of the gap, and the phenotype conferred to cells by the met17-s mutation. (A) The hatched box indicates the 1.5-kb ORF (arrow, start codon; *, stop codon). MET17 plasmids to be used as substrates in the gap repair assay were digested with BspEI and EcoNI to produce a 238-bp gap, indicated by a black box. The plasmid and chromosomal DNA donor sequences contain a nonsense mutation that destroyed a SnaBI site (met17-s) 216 bp downstream of the EcoNI site. (B) The gap produced by BspEI-EcoNI digests consists of noncomplementary 5' overhangs that overlap in one nucleotide (C · C) and is expected to provide a poor substrate for ligation. However, degradation or melting of the EcoNI end could provide microhomologies (C · G and/or GG or CC) for annealing to the overhang produced by BspEI digestion. (C) The first sequence shows the SnaBI site in the MET17 allele; the second line of sequence indicates the A insertion at the SnaBI site, which creates a stop codon and disrupts the recognition site for SnaBI. The met17-s mutation confers to cells a dark brown phenotype when grown on medium supplemented with lead (Pb).

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Rationale of the gap repair assay. (A) The double-strand gap in MET17 on either ARS (open square), CEN ARS (open square and open circle), or integrating (NON) plasmid is repaired from homologous chromosomal or plasmid met17-s sequences. (B) Repair of gapped ARS plasmids without a crossover produces a repaired MET17 plasmid and an unchanged donor sequence (chromosome or plasmid); repair associated with a crossover results in an integrated ARS plasmid. Repair of a gapped CEN ARS plasmid has to occur by a noncrossover mechanism because integration results in a dicentric chromosome or plasmid, which is inviable. Repair of the gapped plasmid that contains no ARS element has to occur by integration to yield a stable transformant. The products were drawn based on the assumption that the gap in the plasmid is not extended by nucleases over the MET17 SnaBI site. (C) The products expected from gap repair unassociated and associated with crossover were distinguished by monitoring the selective and colony color phenotypes conferred by the URA3 and MET17 markers to the recombinants and by the mitotic stability phenotype of these markers. For the identification of single patches by numbers, a grid is included in C6. Patches on independent plates are shown; note that plates 4 and 5 are not from the same master plate. Confluent growth on 5-FOA medium indicated that the Ura+ phenotype was mitotically unstable (i.e., C1 no. 2, and C2, no. 2). Secondary pop-out recombination between duplicated MET17 alleles delineating URA3 leads to the formation of papillae on 5-FOA due to the excision and loss of URA3 (i.e., C1, no. 8 and C3, no. 2). Cells displaying a dark brown phenotype when grown on lead (Pb) plates indicated that the Met+ phenotype was unstable in cells transformed with ARS plasmid (i.e., C4, no. 10) or absent (i.e., C5, no. 2). During further incubation of plate C5 for 4 to 5 days at room temperature, patches that were previously white turned a beige color and showed dark brown papillae, indicating the progressive loss of CEN ARS plasmids (i.e., C5, no. 8). White cells were diagnostic for a stable Met+ phenotype (i.e., C4 no. 2, and C5, no. 46 and 50).

Classification of gap repair products. Genetic and physical tests were used to determine the distribution of gene conversion events associated or unassociated with a crossover. The gap repair products were grouped into categories based on the selective and mitotic stability phenotypes conferred to the recombinants by the marker genes. The Met⁺ Ura⁺ clones from the gap repair assay could have resulted from two different classes of recombination. (i) Repair of the gapped ARS plasmid by gene conversion without a crossover leads to an unstable Ura⁺ Met⁺ phenotype (Ura^+u Met^+u) due to loss of the plasmid under nonselective growth conditions. (ii) Repair of the plasmid followed by integration at the chromosomal met17-s locus results in a stable Ura⁺ Met⁺ phenotype (Ura^+s Met^+s). Transformed gapped CEN ARS and nonreplicating plasmids are constrained to remain episomal or to integrate into the genome during gap repair. Therefore, the majority of Met⁺ transformants from the CEN ARS plasmid were expected to be Ura^+u Met^+u whereas only Ura^+s Met^+s should be detected in assays with nonreplicating plasmids. This separation of phenotypes allowed us to monitor exclusively either gene conversion events unassociated with crossing over or conversion associated with crossing over (integration). The classification of products based on stability of the markers was confirmed by Southern blot analysis. The nonsense mutation in the met17-s allele, used as the DNA donor, has destroyed a SnaBI restriction enzyme site in MET17 and can be used to distinguish between plasmid and chromosomal alleles.

Gap repair proficiency of rad51, rad52, rad53, rad57, rad59, and rad51 rad59 mutants. Initially, the gapped ARS plasmid was used to examine the role of plasmid versus chromosomal sequences as donors in gap repair and the dependence of plasmid-by-plasmid and plasmid-by-chromosome gap repair on RAD genes. In Rad⁺ cells, the efficiency of gap repair using the plasmid and chromosomal donors was comparable (Table 2). The gap repair frequencies were substantially reduced in hosts containing rad51, rad52, rad57, and rad59 mutations compared to wild-type cells, independent of the origin of the donor DNA (Table 2). The rad52 mutant showed the greatest decrease in gap repair (>500-fold), the rad51 and rad57 mutants showed an intermediate decrease (67- to 110-fold), and the rad59 strain showed a 21- to 57-fold decrease. Although not analyzed in detail, a rad55 strain showed a similar reduction in gap repair efficiency to the rad51 and rad57 strains. rad55 and rad57 mutants showed more severe DNA repair defects at low temperatures; however, the defect in gap repair was apparent even at 30°C, the temperature used for the plasmid gap repair assay (16, 20, 26). In the rad53-21 mutant, a moderate two- to fourfold reduction was observed, indicating a possible role of this DNA damage checkpoint gene in the regulation of gap repair. The epistatic relationship between RAD51 and RAD59 for gap repair was also assessed. The gap repair frequency in rad51 rad59 double mutants was synergistically reduced compared to that observed in rad51 or rad59 single mutants. The transformation frequency of the rad51 rad59 double mutants only slightly deviated from that observed in either single mutant alone. The highest reduction in the efficiency of transformation was observed in rad52 mutants (0.16 × 10⁵ CFU/µg of DNA), as reported previously (38).

Gap repair events are biased toward noncrossover products. In the previous experiment, Met⁺ Ura⁺ transformants were selected to determine the frequency of gap repair to restore the MET17 gene. To determine all possible modes of plasmid repair, Ura⁺ transformants were selected and then analyzed for the Met phenotype as well as mitotic stability. For example, Ura^+s Met^+s (integration) recombinants were distinguished from Ura^+u Met^+u (nonintegration) by growth on nonselective medium followed by replica plating either to medium supplemented with 5-FOA or to Pb plates (see Materials and Methods). As shown in Fig. 2, Ura^+u Met^+u cells formed dense patches on 5-FOA plates and cells grown on Pb-plates showed a dark brown colony color phenotype diagnostic for the loss of the MET17 gene. Secondary pop-out recombination events in Ura^+s Met^+s cells would be indicated by the formation of papillae on 5-FOA plates due to the excision and loss of the integrated plasmid URA3 marker by recombination of flanking homologous sequences.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Structural analysis of Ura+ transformants. Yeast DNA isolated from Ura+ transformants was digested with BamHI (B) and SnaBI (S), and Southern blot analysis was performed with a MET17 DNA fragment as the hybridization probe. (A) Schematic representation (not drawn to scale) of the expected DNA repair product to generate an unstable (u) Ura+ Met+ phenotype; also shown is the chromosomal met17-s allele. The sizes of DNA fragments that hybridize to the probe are shown. The lower panel shows a Southern blot of this class of events. (B) Schematic representation of integration events to produce a stable Ura+s Met+s phenotype. The three simplest classes are shown, although multiple integration events to produce fragments of 1.5 and/or 7.2 kb also occur. The lower panel shows a representative Southern blot of this class of events. (C) Schematic representation of events to produce a Ura+u Met phenotype. Conversion of the plasmid MET17 to met17-s is monitored by the appearance of a 7.2-kb fragment. A 1.3-kb BamHI-SnaBI fragment that hybridizes to the probe is diagnostic for a nonhomologous end joining of the gapped plasmid substrate. For both classes, the 6.7-kb met17-s allele is unchanged. Events from a wild-type strain (RAD) are shown on the left Southern blot, and those from a rad51 strain are shown on the right. (D) Schematic representation of an integration event to produce a stable Ura+s Met phenotype. The 9.9- and 3.0-kb fragments are diagnostic of two copies of met17-s; multiple integration results in an additional fragment of 7.2 kb. Southern blots of DNA from RAD and rad51 strains are shown below the schematic. Included as size markers (M) are SnaBI-BamHI-digested plasmid pSB110 (MET17) and genomic DNA, isolated from an untransformed tester strain (met17-s), which produce signals of 1.5 and 6.7 kb, respectively. Fragment sizes are given in kilobase pairs on the left and were determined relative to HindIII-digested lambda DNA run as a standard.

Gap repair of the CEN/ARS and nonreplicative plasmids. To further determine whether the RAD51 pathway is involved in only a particular noncrossover pathway, CEN ARS and nonreplicating plasmids were used as substrates for gap repair. These plasmids are constrained to remain episomal (CEN ARS) or to integrate (nonreplicating plasmid) during gap repair. If the hypothesis that the RAD51 pathway is required only for noncrossover events is correct, we would expect to obtain no Met⁺ transformants in the rad51 and rad57 strains with the CEN ARS plasmid and wild-type levels of Met⁺ transformants with the nonreplicating plasmid. The gap repair frequency from the chromosomal donor with the CEN ARS plasmid was lower than observed with the ARS plasmid (17 × 10²) and was reduced further when the nonreplicative plasmids were used in wild-type strains (8.2 × 10²) (Table 4). In contrast to our expectations, there was a greater reduction in gap repair when the nonreplicative plasmid rather than the CEN/ARS plasmid was used in all of the rad mutants tested. Thus, crossing over is dependent on RAD51, RAD57, and RAD59. As found for the ARS plasmid, gap repair of the CEN/ARS and nonreplicative plasmids was defective in these mutants when using either chromosomal or plasmid templates (Table 4).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

An assay for plasmid gap repair during yeast transformation was developed to distinguish between homologous recombination events resulting in crossover (integration) or noncrossover products by genetic methods. The assay is based on the repair of a gap within a plasmid-borne MET17 gene by a chromosomal or plasmid template containing a nonsense mutation near the 3' end of the MET17 ORF. A series of plasmids was constructed that contain the selectable/counterselectable marker URA3 and the colony color marker MET17 but differ in their ability to replicate in yeast. These features can be used to distinguish between crossover and noncrossover modes of gap repair by growth on various media (Fig. 2). The goals in the establishment of this assay were to assess the relationship between gene conversion and crossing over and to determine the basis for RAD51-independent mitotic recombination events. Several hypotheses have been put forward to explain RAD51-independent events. First, Sugawara et al. (60) suggested that RAD51, RAD54, and RAD57 are not directly involved in recombination but instead are required to facilitate access to chromatin templates. Second, Rattray and Symington (45) proposed that RAD51, RAD54, RAD55, and RAD57 are required for gene conversion but play a less important role in events that can be resolved as crossovers. As described in detail below, our results are inconsistent with either hypothesis. Instead, our results support the idea that strand invasion or strand annealing between repeated sequences can occur in the absence of RAD51.

RAD51 is required for recombination from chromosomal or plasmid donors. Using the ARS plasmid substrate, the gap repair frequency was reduced 98-fold with a chromosomal template and 110-fold with a plasmid template in rad51 strains (Table 2). Similar reductions were observed in rad57 mutants, consistent with other studies indicating that these genes function in the same pathway. This pattern was also evident with the CEN/ARS and integrating vectors (Table 4). Therefore, in this DSB repair system, RAD51 is required for repair even when the donor locus is expressed and on a plasmid. This contrasts with the results of Sugawara et al. (60), who found efficient repair of DSBs from expressed plasmid donor templates in rad51 mutants but a requirement for RAD51 when the donor was chromosomal or transcriptionally silent and on a plasmid. Although in the plasmid gap repair system described here the recipient sequences are in the form of naked transformed DNA, the donors are in the same configuration as those used in the study by Sugawara et al. (60). Thus, donor accessibility is expected to be similar in the two systems. We cannot exclude the possibility that RAD51 plays an additional role in protection of the broken ends and that this role is more important when the recipient DNA is naked instead of in chromatin.

MAT-inc

MAT-inc

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Model for RAD51-independent recombination of inverted repeats. Following introduction of a DSB in one of the repeats, one end is resected to produce a 3' single-stranded tail that invades the other repeat, possibly through the action of Rad52. DNA synthesis is primed from the invading strand and proceeds to the end of the DNA molecule (the other side of the break). The linear molecule formed contains short sequences corresponding to the 5' end of the inverted repeat at its ends. If the linear intermediate is degraded by a 5'-3' exonuclease so that complementary single-stranded regions are revealed, then strand annealing can occur to form two types of products. One has the same structure as a reciprocal exchange, and the other has the same structure as the parental plasmid. The inverted repeats are shown by thick arrows, DNA synthesized during repair is shown by dashed lines, and sequences between the inverted repeats are designated A and B.

The RAD51-dependent pathway is biased toward noncrossover products. Previous studies of spontaneous mitotic recombination with an ade2 inverted repeat recombination substrate led to the suggestion that the RAD51 pathway (including RAD54, RAD55, and RAD57) is primarily required for gene conversion. In this study, DSBR events associated or unassociated with crossing over were differentiated in two ways. First, repair of the ARS plasmid substrate results in integration (crossover) or the plasmid remains episomal (noncrossover). These two classes can be distinguished by genetic and physical tests. Second, repair of the nonreplicative plasmid can occur only by integration, and repair of the CEN ARS plasmid to yield stable transformants can occur only by a noncrossover mechanism. In wild-type strains, repair of the ARS plasmid yielded 76% episomal and 21% integration events (Table 3). While this is different from the 50% integration reported by Orr-Weaver and Szostak (37), it is consistent with recent studies of gap repair in S. cerevisiae and in other organisms (12, 35, 39, 44). Studies in Drosophila and U. maydis have led to the proposal of the synthesis-dependent strand annealing (35) and migrating D-loop (12) models to explain the low incidence of crossing over associated with DSBR. In these models, strand invasion and DNA synthesis primed from the invading 3' end occur as described for the DSBR model, but instead of forming a Holliday junction intermediate, the invading strand is displaced and can then pair with the single-stranded tail on the other side of the break (Fig. 5). Crossover events can occur if the displacement loop pairs with the other side of the break, or by appropriate strand cleavages such that a Holliday junction is formed. To account for the level of crossing over observed in this study, we suggest that the strand invasion intermediate is converted to a double Holliday structure 40% of the time. The differences in the level of associated crossing over found in different studies could reflect locus-specific effects on the ratio of these intermediates.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Models for the recombinational repair of DSBs. (A) The DSBR model. After formation of a DSB, the 5' ends are resected to form 3' single-stranded tails. One of the 3' single-stranded tails invades a homologous duplex and primes DNA synthesis. The displaced strand from the donor duplex pairs with single-stranded DNA at the other side of the break and is the template for DNA synthesis. After ligation, a double Holliday structure is formed and can be resolved to yield noncrossover or crossover products. (B) The synthesis-dependent strand-annealing (SDSA)/migrating D-loop model. The first two steps are the same as those in the DSBR model, but most of the time a double Holliday junction intermediate is not formed. Instead, the invading strand that has been extended by DNA synthesis is displaced from the donor duplex and can anneal to the single-stranded tail on the other side of the break. The resulting gaps are filled by DNA synthesis and ligation to yield a noncrossover product. To account for the crossover products recovered from plasmid gap repair, we propose that 40% of the events form a Holliday junction intermediate and, of these, 50% resolve to generate crossover products. Dashed lines represent newly synthesized DNA.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Models for inversions between ade2 inverted repeats (9, 46). (A) In G2 cells, the inverted repeats on different chromatids (open arrows) can pair such that the intervening DNA sequences (shown as solid arrows) are in an antiparallel configuration. A gene conversion event initiated within one repeat that extends to the other repeat could result in the inversion of the intervening DNA. (B) A reciprocal crossover between inverted repeats located on the same chromatid inverts the intervening DNA sequences, but the product is indistinguishable from a G2 conversion event. The inverted repeats are shown by open boxes with arrowheads, the short repeat corresponds to a truncation of the 5' end, and the long repeat contains a point mutation shown by the vertical line.

Aberrant events in rad51 mutants. The proportion of repair events associated with crossing over was increased in rad51 and rad57 mutants compared to the wild-type strain. Although this difference was not significant for the Met⁺ events, the Met events showed a significant bias toward integration of the plasmid. Eighty percent of the Ura⁺ Met transformants that occurred by recombinational repair of the ARS plasmid in the rad51 strain were the result of integration of the plasmid and duplication of the met17-s allele. A similar alteration in the ratio of crossover to noncrossover recombinants was observed during plasmid gap repair in rad51 mutants of U. maydis (13). Although crossover events were more frequently recovered when the ARS plasmid was used, integration of the nonreplicative plasmid was reduced more than 100-fold in the rad51 mutant (Table 4), indicating an important role for RAD51 in reciprocal exchange. The rare crossover events that occur in rad51 mutants could potentially occur by two sequential break-induced replication events primed from the plasmid ends to duplicate the met17-s locus and both chromosome arms (27, 33).

Gap repair is partially dependent on RAD53. RAD53 is classified as a member of the RAD52 epistasis group but is thought to affect recombination indirectly by transducing signals induced by DNA damage and stalled replication complexes to downstream targets (2, 61, 68). RAD53 encodes an essential, cell cycle-regulated protein kinase. rad53-21 is a conditional mutation that confers defects in the replication checkpoint and damage-induced phosphorylation of the Dun1 protein kinase (2). Consistent with a previous study (14), we found a small (two to fourfold) reduction in the efficiency of gap repair in the rad53-21 strain. However, recombination occurred at much higher frequencies than were observed for the other rad mutants, suggesting that phosphorylation of Rad proteins by Rad53/Spk1 is unlikely to play a significant role in recombinational repair.

Plasmid gap repair is dependent on RAD52. Repair of the gapped ARS plasmid occurred at very low frequency in the rad52 strain, and no products were obtained that resulted from recombinational repair. This is consistent with other assays for mitotic recombination, which have shown a strong dependence on RAD52 function, and with our suggestion that RAD52 is an essential component of RAD51-dependent and RAD51-independent recombination pathways (3, 4).

RAD59 is required for RAD51-dependent and RAD51-independent recombination. RAD59 encodes a Rad52 homologue and was identified by its requirement for RAD51-independent recombination using an ade2 inverted-repeat substrate (4). A synergistic decrease in spontaneous recombination of chromosomal inverted repeats was observed in rad51 rad59 double mutants. In this study, we show a defect in repair of the gapped ARS plasmid in rad59 mutants and a synergistic decrease in rad51 rad59 double mutants. The observation that 95% of repair events require RAD59 suggests that Rad59 plays an important role in the RAD51 pathway, possibly as an accessory factor for Rad52. The synergistic decrease in gap repair efficiency observed in the rad51 rad59 double mutant is consistent with observations obtained with the ade2 inverted repeat and indicates an important role for RAD59 in both spontaneous and DSB-induced recombination. Ninety-nine percent of the repair events involving the gapped ARS plasmid require RAD51, indicating that most repair events are mediated by the RAD51 pathway. However, some repair can occur in the absence of RAD51, and these residual events require RAD52 and RAD59. This observation supports the hypothesis that Rad52 or the Rad52-Rad59 complex promotes strand invasion in vivo, albeit inefficiently. This type of recombinational repair might be initiated by annealing of transiently single-stranded regions of the substrates. Such events are likely to be favored during replication or by transcriptional activation of sequences in close proximity, such as repeats. The idea that proteins without RecA homology can catalyze strand invasion is not without precedent. The bacteriophage lambda  protein is important for lambda recombination and catalyzes strand annealing and strand exchange in vitro (22, 24). Recent studies have shown that  protein and RecT, a homologue of  protein, can promote recA-independent gene replacement in E. coli, suggesting a role in strand invasion in vivo (71, 72).  protein forms ring structures on DNA similar to those formed by the yeast and human Rad52 proteins (41, 56, 67). The observation that Rad52 catalyzes single-strand annealing in vivo and in vitro is consistent with the structural analogy to lambda  protein (34, 59). Thus it seems reasonable to propose that Rad52, or the Rad52/Rad59 heterodimer catalyze an alternate pathway for strand invasion in vivo. However, this appears to be inefficient under most circumstances.