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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Radiat Res. Author manuscript; available in PMC Aug 1, 2010.
Published in final edited form as:
Radiat Res. Aug 2009; 172(2): 141–151.
doi:  10.1667/RR1675.1
PMCID: PMC2741414
NIHMSID: NIHMS136836

Rad1, rad10 and rad52 Mutations Reduce the Increase of Microhomology Length during Radiation-Induced Microhomology-Mediated Illegitimate Recombination in Saccharomyces cerevisiae

Abstract

Illegitimate recombination can repair DNA double-strand breaks in one of two ways, either without sequence homology or by using a few base pairs of homology at the junctions. The second process is known as microhomology-mediated recombination. Previous studies showed that ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated recombination in trans during rejoining of unirradiated plasmids or during integration of plasmids into the genome. Here we show that radiation-induced microhomology-mediated recombination is reduced by deletion of RAD52, RAD1 and RAD10 but is not affected by deletion of RAD51 and RAD2. The rad52 mutant did not change the frequency of radiation-induced microhomology-mediated recombination but rather reduced the length of microhomology required to undergo repair during radiation-induced recombination. The rad1 and rad10 mutants exhibited a smaller increase in the frequency of radiation-induced microhomology-mediated recombination, and the radiation-induced integration junctions from these mutants did not show more than 4 bp of microhomology. These results suggest that Rad52 facilitates annealing of short homologous sequences during integration and that Rad1/Rad10 endonuclease mediates removal of the displaced 3′ single-stranded DNA ends after base-pairing of microhomology sequences, when more than 4 bp of microhomology are used. Taken together, these results suggest that radiation-induced microhomology-mediated recombination is under the same genetic control as the single-strand annealing apparatus that requires the RAD52, RAD1 and RAD10 genes.

INTRODUCTION

Chromosomal DNA double-strand breaks (DSBs) are highly cytotoxic to cells. Homologous recombination (HR) is the main mechanism for repair of DSBs in yeast. In the presence of a homologous sequence, the DSB is repaired by the gene conversion pathway that involves strand invasion and copying of the genetic material from the homologous template (1). When a DSB is flanked by two direct repeats, single-strand annealing occurs and results in deletion of one of the repeats and all of the intervening sequences (2). The HR process is mediated by the RAD52 epistasis group (3).

Illegitimate recombination or nonhomologous end joining (NHEJ) joins broken DNA ends without extensive sequence homology (4). It is a minor DSB repair pathway in yeast that is evident in the absence of essential genes in homologous recombination or in the absence of homologous sequences (57). The main genetic factors required for NHEJ in yeast include the yKu70/yKu80 heterodimer, Rad50/Mre11/Xrs2 complex and Dnl4/Lif1/Nej1 complex (8).

An alternative pathway of end joining uses a few base pairs of homology at the recombined junctions and is called microhomology-mediated recombination (MHMR) (5, 6). MHMR is independent of YKU70 and YKU80 and thus is distinct from the classical NHEJ pathway (7, 912). MHMR is usually associated with deletions and other genomic rearrangements that have been implicated in human cancer and genetic diseases (1317).

Ionizing radiation and restriction enzymes increase the frequency of nonhomologous integration (NHI) by severalfold in yeast (11, 18, 19). Previously, we found that ionizing radiation- and restriction enzyme-induced NHI events exhibited an increased frequency of microhomology use at the integration junctions (20). We also showed that restriction enzymes enhance the efficiency of NHI at non-restriction sites and that overexpression of I-SceI endonuclease induces MHMR events at random genomic locations rather than being targeted to the proximity of the I-SceI-induced DSB. We further demonstrated that exposure of yeast and mammalian cells to radiation before transformation of the linearized plasmid increases the frequency of microhomology-mediated end joining (MMEJ) of the unirradiated plasmid that is in trans to radiation-induced damage (21). These previous results suggest that DSBs induce MHMR in trans at random non-targeted sites during integration of the plasmid into the genome. However, little is known about the genetic control of the radiation-induced MHMR pathway. Since MHMR is independent of Ku (7, 912), we investigated some candidate genes other than those involved in the classical NHEJ pathway in the current work. We examined the following genes whose encoded products exhibit homologous pairing or nuclease activities, which we assumed may play a role in the radiation-induced MHMR pathway.

The RAD51 and RAD52 genes are involved in DSB repair in yeast. Both gene products promote strand exchange and annealing of complementary DNA strands in homologous recombination (22, 23). Rad52 displaces RPA from single-stranded DNA and promotes assembly of Rad51 onto single-stranded DNA to form nucleoprotein filaments for strand invasion (23, 24). RAD52 also promotes RAD51-independent single-strand annealing in the presence of two direct repeats flanking a DSB (25).

The Rad1/Rad10 complex in S. cerevisiae functions as a structure-specific single-stranded endonuclease that cleaves at the duplex/3′ single-strand DNA junction (26). This complex is required for removal of 3′ single-strand DNA from the recombining DNA in single-strand annealing (27) and for 5′ incision of UV-radiation-induced damage during nucleotide excision repair (28). Rad2, a 5′ to 3′ exonuclease and a 5′ flap endonuclease (29, 30), is required for 3′ incision of UV-radiation-induced lesions in nucleotide excision repair (31).

In this study, we examined the role of the potential genes RAD51, RAD52, RAD1, RAD10, RAD2 and DNL4 in MHMR in the absence and presence of γ radiation by sequence analysis of the integration target sites. We examined the effect of these gene mutations on the frequency of spontaneous and radiation-induced NHI, and in particular on the distribution of microhomology use during nonhomologous integration.

MATERIALS AND METHODS

Strains and Media

Experiments were performed in the haploid Saccharomyces cerevisiae strain RSY12 (MATa leu2-3,112 his3-11,15 ura3Δ::HIS3), in which the entire URA3 open reading frame and promoter sequence was replaced by the HIS3 gene (19). rad51, rad52 and rad1 derivatives of the RSY12 background were described previously (32). The rad10 derivative was constructed by integration of pDD37 into RSY12 (33). The disruption cassettes of RAD2 and DNL4 containing a LEU2 marker were generated by PCR-based methods using the plasmid YEplac128 as the template and the following primers: RAD2-LEU2-FW 5′-GATGTAATA ACATATACT TATGTATGATTTAC-TATTTATAAACATTACCTACCGTCATCACCG AAACG-3′, RAD2-LEU2-RC 5′-GTAAGAGAATTTCAACCGCAACAGG-TAAA CTAAAGAAAAGA AAGATGTAAATAGGCGTATCAC-GAGGC-3′, DNL4-LEU2-FW 5′-TAGGTATGATATCAGCAC-TAGATTCTATACCCGAGCCCCAAAACTTTGCG ACCGTCA-TCACCGAAACG-3′ and DNL4-LEU2-RC 5′CACCATCAG-TAGTTGA CTACGGGGAAGTC TTCTTCAGGCACTTGA-CAGTTATAGGCGTATCACGAGGC-3′. The disruption cassette was then transformed into RSY12 to generate the rad2 and dnl4 derivatives. E. coli strain DH5α was used for the maintenance and amplification of plasmid DNA.

Plasmids

YEplac195 contains the URA3 marker for selection and the 2-μm origin of replication and was used to control for transformation efficiency. Plasmid pM151 was described previously (19) and contains a 1.1-kb URA3 HindIII fragment.

Molecular Techniques

Standard methods were followed except as noted below. pM151 was digested with BglII and prepared as described (19). For yeast transformation, the “Lithium Acetate/Single Stranded DNA/PEG” transformation method was used (34). The spontaneous and radiation-induced integration target sites were determined by direct genomic sequencing or plasmid rescue that was described previously (20).

γ Irradiation

After transformation with the integrating DNA, yeast cells were plated onto synthetic complete medium lacking uracil and exposed to 50 Gy of γ rays using a Mark-I irradiator (J. L. Shepherd and Associates, San Fernando, CA), with a 137Cs source and a dose rate of 5.29 Gy/min.

Statistical Analysis

The frequency of NHI before irradiation was compared to that after irradiation by the Student’s t test. The frequencies of microhomology use during spontaneous and radiation-induced NHI were analyzed by a χ2 test, Fisher’s exact test or Mann-Whitney U test. A χ2test with Yates’ correction was used when one of the expected frequencies was less than 5 in a 2 by 2 table.

RESULTS

Radiation-Induced Increase of Microhomology Length during Nonhomologous Integration is Reduced by rad52 but not rad51 Deletions

We determined the roles of the RAD51 and RAD52 genes, which are known to be involved in homologous pairing (22, 23), in spontaneous and radiation-induced NHI. We transformed the BglII-linearized URA3-containing plasmid pM151 and the circularized episomal plasmid YEplac195 in parallel, as the transformation efficiency control, into the mutant cells in the absence and presence of 50 Gy of γ radiation and selected for stable Ura+ transformants. Genomic DNA was subsequently isolated from stable transformants. The junctions between the integrated plasmid and the flanking genomic sequences were recovered by plasmid rescue and sequenced. The sequences obtained were compared with the yeast chromosomal DNA in the Saccharomyces Genome Database (SGD) to identify the integration target sites. The rad51 and rad52 mutations caused a 2.0-and 1.8-fold decrease, respectively, in the frequency of spontaneous NHI events relative to wild-type cells (P < 0.005 for rad51, not significant for rad52; Table 1). Radiation increased the frequency of NHI events by 4.7-fold in the wild-type, 14-fold in the rad51 mutant (P < 0.01), and 70-fold in the rad52 mutant (P < 0.01) (Table 1). In the rad51 mutant, six out of 16 junctions (38%) displayed 2 to 3 bp of microhomology in spontaneous integration (Figs. 1B and and2A),2A), whereas after irradiation 21 out of 28 NHI events (75%) involved 2 to 5 bp of microhomology, showing an induction of microhomology use after irradiation (P < 0.05) (Fig. 2B). Nine out of 28 junctions in irradiated rad51 mutant displayed 4 bp or more of microhomology, which is not significantly different from the distribution of microhomology use in irradiated wild-type cells (20), namely nine out of 22 junctions with 4 bp or more by a χ2 test (Fig. 1A). There was also no significant difference in the overall distribution of microhomology use between irradiated rad51 mutant and irradiated wild-type cells by the Mann-Whitney test. These results indicate that Rad51 does not play any role in radiation-induced MHMR.

FIG. 1
The distribution of microhomology use in spontaneous and radiation-induced NHI events in (panel A) wild-type strain RSY12 [data from ref. (20)] and is shown here for comparison. Panel B: rad51 and (panel C) rad52 mutants, in which 10 Gy to the rad52 mutant ...
FIG. 2
Target sequences of NHI events in rad51 mutant. Panel A: Spontaneous events; panel B: after exposure to radiation. The genomic location, locus and gene of the target sites are shown in the Supplementary Material, Table S1.
TABLE 1
Relative Frequency of NHI Events

Similarly, in the rad52 mutant, two out of 18 spontaneous NHI events (11%) used 2 bp of microhomology (Figs. 1C and and3A)3A) whereas 10 of 22 NHI events (45%) displayed 2 to 5 bp of microhomology in mutant cells irradiated with 50 Gy (Fig. 3B), showing an induction of microhomology use after irradiation (P < 0.05). Noticeably, irradiated rad52 mutant cells exhibited fewer MHMR events with ≥4 bp of microhomology compared to irradiated wild-type cells, namely only two out of 22 junctions (9%) in irradiated rad52 mutant cells compared to nine of 22 junctions (41%) in irradiated wild-type cells (P < 0.05, χ2 test). Furthermore, there was a significant difference in the overall distribution of microhomology use, in which no cut-off value for microhomology is required, between the rad52 mutant irradiated with 50 Gy and the irradiated wild-type cells (P < 0.05, Mann-Whitney test).

FIG. 3
Target sequences of NHI events in rad52 mutant. Panel A: Spontaneous events; panel B: after exposure to 50 Gy of γ radiation; panel C: after exposure to 10 Gy of γ radiation. The genomic location, locus and gene of the target sites are ...

Because the rad52 mutant is highly sensitive to ionizing radiation (35), the phenotype of the rad52 mutant cells after exposure to 50 Gy of radiation could be confounded by selection bias of the mutant cells that survived after irradiation. To exclude this possibility, we transformed the integrating DNA into the rad52 mutant and exposed cells to 10 Gy of γ rays, a dose equitoxic to 50 Gy in the wild-type cells that results in about 45% survival (data not shown). Exposure to 10 Gy of γ rays increased the frequency of NHI by 15-fold in the rad52 mutant (P < 0.01) (Table 1). In this instance, eight out of 16 (50%) radiation-induced NHI events contained 2 to 4 bp of microhomology (Fig. 1C and and3C),3C), showing a significant induction of microhomology use after irradiation with 10 Gy (P < 0.05). In particular, one out of 16 NHI events with ≥4 bp of microhomology were observed in the rad52 mutant cells irradiated with 10 Gy. This is significantly different from nine out of 22 events in the irradiated wild-type cells (P < 0.05, χ2 test), indicating that the NHI events in the rad52 mutant irradiated with 10 Gy exhibited less extensive microhomology. The overall distribution of microhomology use in the rad52 mutant irradiated with 0 Gy was also significantly different from that of the irradiated wild-type cells (P < 0.05, Mann-Whitney test). The fraction of MHMR events in the rad52 mutant cells irradiated with 10 Gy was similar to that of the mutant cells irradiated with 50 Gy (50% for 10 Gy compared to 45% for 50 Gy). These observations eliminate the possibility that the rad52 phenotype was due to selection of a small population of rad52 mutant cells that survived after irradiation. These results indicate that radiation-induced MHMR with 4 bp or more is required by RAD52.

Human Rad52 has been shown to bind DSBs and to protect DNA ends from exonuclease attack in vitro (36). Our previous results have shown that radiation protects the single-stranded termini of the integrating DNA from degradation and that the single-stranded termini are required for MHMR (20). We determined whether degradation of the PSS DNA ends may have caused the lower extent of microhomology use in irradiated rad52 mutant cells. In the unirradiated rad52 mutant, 18 out of 72 nucleotides were deleted at the PSS ends of the integrating DNA whereas only nine out of 88 were deleted in the rad52 mutant cells after 50 Gy of radiation (P < 0.05), indicating that there is a radiation-induced protection of DNA termini in the rad52 mutant cells as in wild-type cells (20). This rules out the possibility that the lower extent of microhomology use in irradiated rad52 mutant than in the wild-type cells was caused by degradation of the DNA ends that provide the microhomologies prior to integration.

Radiation-Induced Recombination with Microhomology of more than 4 bp is Reduced by Mutation of the RAD1 and RAD10 Genes

We examined the roles of the RAD1 and RAD10 genes in spontaneous and radiation-induced NHI and found that the rad1 and rad10 mutations did not have any effect on the frequency of spontaneous NHI events (Table 1). Radiation increased the frequency of NHI by an average of 2.55-fold in the rad1 mutant (P < 0.05) and by 2.61-fold in the rad10 mutant cells (P < 0.005) (Table 1). In the rad1 mutant, four out of 14 (29%) spontaneous NHI events displayed 2 to 3 bp of microhomology whereas 17 out of 24 (71%) radiation-induced NHI events involved 2 to 4 bp of microhomology (Figs. 4A and and5).5). This demonstrates an increase of microhomology use after irradiation (P < 0.05, χ2 test). Of note, the extent of microhomology use in the irradiated rad1 mutant was significantly lower than that observed in the wild-type cells, where none of 24 junctions in the irradiated rad1 mutant displayed more than 4 bp, compared to four out of 22 radiation-induced events in the wild-type cells (P < 0.05, Fisher’s exact test). In the rad10 mutant, four out of 20 (20%) junctions in spontaneous NHI events displayed 2 to 3 bp of microhomology whereas 13 out of 24 (54%) radiation-induced NHI events in the wild-type cells used 2 to 4 bp of microhomology (P < 0.05, χ2 test) (Figs. 4B and and6).6). Similar to the irradiated rad1 mutant, none of 24 junctions in the irradiated rad10 mutant used more than 4 bp of microhomology, which again is significantly different from that of irradiated wild-type cells (P < 0.05, Fisher’s exact test). These results led us to conclude that the Rad1/Rad10 complex is required for radiation-induced MHMR involving more than 4 bp of microhomology.

FIG. 4
The distribution of microhomology use in spontaneous and radiation-induced NHI events in (panel A) rad1, (panel B) rad10 and (panel C) rad2 mutants.
FIG. 5
Target sequences of NHI events in rad1 mutant. Panel A: Spontaneous events; panel B: after exposure to γ radiation. The genomic location, locus and gene of the target sites are shown in Table S3.
FIG. 6
Target sequences of NHI events in rad10 mutant. Panel A: Spontaneous events; panel B: after exposure to γ radiation. The genomic location, locus and gene of the target sites are shown in Table S4.

Radiation-Induced MHMR is not Reduced by Mutation in the RAD2 Gene

To determine the effect of a nucleotide excision repair function in addition to the Rad1/Rad10 endonuclease complex in radiation-induced MHMR, we examined whether Rad2, a structure-specific endonuclease required for nucleotide excision repair that cleaves 5′ flap structures (30), was involved. The rad2 mutation did not have any effect on the frequency of spontaneous NHI (Table 1). Radiation increased the frequency of NHI events by 6.6-fold in the rad2 mutant (P < 0.005) (Table 1). Six out of 18 (33%) spontaneous NHI events in the rad2 mutant involved 2 and 3 bp of microhomology whereas 11 out of 16 (69%) of radiation-induced NHI events contained 2, 3, 4 and 7 bp of microhomology (Figs. 4C and and7),7), showing a significant induction of microhomology use after irradiation (P < 0.05). In particular, four out of 16 radiation-induced MHMR events in the rad2 mutant used 4 bp or more whereas none of 18 spontaneous events used more than 3 bp, showing a significant increase in the length of microhomology used in the irradiated rad2 mutant (P < 0.05, Fisher’s exact test), similar to the irradiated wild-type cells (Fig. 1A). In addition, four out of 16 junctions in irradiated rad2 mutant displayed 4 bp or more of microhomology compared to nine out of 22 junctions in irradiated wild-type cells, which is not significantly different (by χ2 test). There was also no significant difference in the distribution of microhomology use between the irradiated rad2 mutant and irradiated wild-type cells by the Mann-Whitney test. These results showed that the rad2 mutation does not reduce radiation-induced MHMR, in contrast to the rad1/rad10 mutants.

FIG. 7
Target sequences of NHI events in rad2 mutant. Panel A: Spontaneous events; panel B: after exposure to γ radiation. The genomic location, locus and gene of the target sites are shown in Table S5.

The dnl4 Mutation Reduces Spontaneous and Radiation-Induced Nonhomologous Integration

To determine whether radiation-induced NHI events are dependent on DNL4, which is required for religation during NHEJ (8), we examined the effect of the dnl4 mutation on NHI in the absence and presence of ionizing radiation. The dnl4 mutant caused an 8.6-fold decrease in the frequency of spontaneous NHI compared to the wild-type cells (P < 0.01) (Table 1). Radiation caused a 4.5-fold increase in the NHI frequency in the dnl4 mutant (P < 0.01), similar to wild-type cells. These results indicate that the radiation-induced increase in the NHI frequency is independent of DNL4. Out of 11 spontaneous NHI events and eight radiation-induced NHI events in the dnl4 mutant, only two junctions from the same NHI transformant, in the irradiated dnl4 mutant, could be sequenced by the plasmid rescue method. These junctions did not involve any microhomology, exhibited deletions from the ends of the integrating plasmid, and involved a 950-bp deletion at the integration target site (data not shown).

DISCUSSION

We showed previously that ionizing radiation and restriction enzymes increase the frequency of MHMR events that are probably in trans at non-targeted sites (20). We report here that radiation-induced MHMR partly requires the RAD52, RAD1 and RAD10 genes but not the RAD51 and RAD2 genes, which is the same for single-strand annealing, suggesting that MHMR resembles the single-strand annealing pathway in its genetic control.

Radiation Increases the Normalized Frequency of both MHMR and Non-MHMR NHI Events in rad51 and rad52 Mutants

Since radiation increased the relative frequency of NHI in all of the mutants we studied (Table 1), we determined the normalized MHMR frequency in the absence and presence of irradiation (Fig. 8A), in which the relative frequency of NHI was multiplied by the fraction of MHMR events (described in Fig. 8). We also determined the normalized frequencies of non-MHMR events (Fig. 8B). In the wild-type cells, radiation caused a 14-fold increase in the normalized frequency of MHMR events and a 1.4-fold increase in that of non-MHMR events (P values shown in Fig. 8). This suggests that radiation specifically induces the MHMR pathway in the wild-type cells. Interestingly, in the rad51 mutant radiation caused a 27-fold and 5.6-fold increase, respectively, in MHMR and non-MHMR events. In the rad52 mutant, 10 Gy caused a 70-fold increase in MHMR events and an 8.7-fold increase in non-MHMR events while 50 Gy caused a 247-fold and 48-fold increase, respectively, in MHMR and non-MHMR events. This indicates that radiation enhances the normalized frequencies of both MHMR and non-MHMR events in the rad51 and rad52 mutants. It is possible that radiation-induced NHI occurs at radiation-induced DSBs or other damaged sites in these mutants. The normalized frequency of non-MHMR events in the rad52 mutant irradiated with 10 Gy was about 4.1-fold higher than that of the irradiated wild-type cells (3.17 compared to 0.770), indicating a specific induction of a non-MHMR NHI pathway in the rad52 mutant cells. It was found previously that the yeast mutant strains deficient in Rad52 and Yku70 are more sensitive to ionizing radiation than the rad52 single mutant strain (7). The hypersensitivity of the rad52/yku70 double mutant could be explained by our finding that a non-MHMR pathway is induced by radiation in the rad52 mutant that might be mediated by Ku and is partly taking over the DSB repair in the absence of Rad52. The normalized frequency of MHMR in the rad52 mutant irradiated with 10 Gy was similar to that of irradiated wild-type cells (3.17 compared to 2.82), indicating that the rad52 mutation does not have any effect on the overall efficiency of MHMR but rather on the length of microhomology relative to events in wild-type cells based on sequencing results.

FIG. 8
The actual effect of each mutation on MHMR and non-MHMR events after normalization with the relative frequency of NHI. Panel A: The normalized frequency of MHMR events. Panel B: The normalized frequency of non-MHMR events. The relative frequency of NHI ...

In contrast, in the rad1 and rad10 mutants, radiation caused a 5.6- and 8.1-fold increase, respectively, in the normalized frequency of MHMR events and did not have much effect on non-MHMR events, similar to the wild-type cells. The normalized frequencies of MHMR in the irradiated rad1 and rad10 mutants were about 2.2-and 2.8-fold lower than that of irradiated wild-type cells; these observations are consistent with the sequencing results indicating that Rad1 and Rad10 are partly required for radiation-induced MHMR. In the rad2 mutant, radiation caused a 12-fold increase in MHMR and the normalized frequency of non-MHMR events before and after irradiation was not significantly different, similar to wild-type cells.

Increase of Microhomology Length during Radiation-Induced MHMR is Reduced by Mutation of rad52

Both yeast and human Rad52 proteins have been shown to promote strand exchange as well as annealing of complementary single-stranded DNA in vitro (22, 37). Our results suggest that Rad52 facilitates microhomology search and annealing of short homologous sequences in radiation-induced MHMR that is likely in trans at non-targeted sites (see model in Fig. 9). Our results are consistent with the observation that fission yeast Rad22, a homolog of the budding yeast Rad52, is required for microhomology-mediated end joining of transformed linearized substrates containing flanking microhomologies (38). A RAD52-dependent pathway of DSB rejoining has been proposed in yeast based on the observations that rejoining of complementary DSBs with overhangs of 8 nucleotides or longer was partially dependent on RAD52 (39). In contrast, our results show that after irradiation, microhomologies of 4 to 7 bp are dependent on Rad52.

FIG. 9
Schematic diagram of the mechanism of spontaneous and radiation-induced MHMR. In unirradiated cells, the majority of spontaneous nonhomologous integration events are mediated by illegitimate recombination that does not use microhomology. In irradiated ...

It has been shown that Rad52 involves only annealing of 3′ overhangs in homologous recombination (1), but our results, in agreement with others (39), imply that Rad52 also involves annealing of short 5′ overhangs. In addition, our results suggest the existence of a Rad52-independent MHMR pathway that has been proposed previously (39, 40). Rad52 is known to play an important role in homologous recombination to repair DSBs (41). It was suggested that either Rad52 or Ku binds to DSBs in which Rad52 directs into homologous recombination whereas Ku initiates the NHEJ pathway (36). Our findings suggest a novel role for Rad52 in promoting the MHMR pathway of end joining; thus Rad52 seems to be able to direct into both homologous recombination and the end-joining pathway. On the other hand, the observation that Rad51 is not required for radiation-induced MHMR indicates that Rad51 is exclusively required for extensive homology annealing in homologous recombination. In fact, the presence of Rad51 prevents the occurrence of recombination between short homologous sequences (42).

Radiation-Induced MHMR Partly Requires RAD1 and RAD10

When more than 4 bp of microhomology are used during NHI, a 3′ single-strand DNA is displaced from the integrating DNA after base-pairing between short homologous sequences of the integrating DNA and the genomic target sequence (Fig. 9). Our results suggest that the Rad1/Rad10 endonuclease is required for removal of the displaced 3′ single-strand DNA end from the integrating DNA during NHI, which is consistent with the observation that the Rad1 protein is involved in 3′ flap removal during microhomology-mediated end joining in the repair of HO-induced DSBs (40). This biochemical activity of Rad1/Rad10 is required to specifically remove 3′ single-strand DNA displaced from the duplex DNA molecules (26), which arises in single-strand annealing (27) and in nucleotide excision repair (28). On the other hand, Rad2, a 5′ flap endonuclease required for 3′ incision of UV-radiation-induced lesions in nucleotide excision repair (30), is not required for radiation-induced MHMR in our experiments. This indicates that the RAD1 and RAD10 genes, and not general nucleotide excision repair genes, are specific for radiation-induced MHMR.

Spontaneous and Radiation-Induced NHI are Dependent on DNL4

Our results showed that the dnl4 mutation decreased the frequency of spontaneous and radiation-induced NHI events to the same extent, suggesting that DNL4 is required for both non-MHMR and MHMR pathways. It is likely that the NHI events in the dnl4 mutant are mediated by CDC9, which encodes the mammalian homologue of ligase I required for DNA replication (42). Two junctions from one irradiated dnl4 mutant were sequenced. These junctions did not involve any microhomology, exhibited deletions from the ends of the integrating plasmid, involved a large deletion at the integration target site, implying that NHI events occur inefficiently in the dnl4 mutant. Since the 4-bp overhangs of the transforming plasmid that are lost in the dnl4 mutant are used for MHMR, it is likely that NHI events in the dnl4 mutant do not involve microhomology. Based on these results, it is likely that radiation-induced MHMR events are dependent on DNL4, which is supported by previous results showing that the dnl4 mutant exhibited a reduced frequency of microhomology-mediated end joining in the repair of HO-induced DSBs in yeast (40).

The Radiation-Induced MHMR Pathway Resembles Single-Strand Annealing

We report here that radiation-induced MHMR partly requires the RAD52, RAD1 and RAD10 genes, which are also involved in single-strand annealing, suggesting that MHMR resembles single-strand annealing in its genetic requirements, and that it may be mediated by part of the single-strand annealing apparatus. Our results are in agreement with a recent study that the fission yeast protein Rad22 (homologue of the budding yeast protein Rad52) and Exo1 are required for microhomology-mediated end joining in fission yeast, and the authors proposed that microhomology-mediated end joining is related to single-strand annealing (38). Our result that radiation-induced MHMR does not require Rad51 further supports this similarity since it also does not play any role in the single-strand annealing pathway. Both MHMR and the single-strand annealing pathway are accompanied by deletions of the intervening sequences between the homologous sequences, and only one of the repeats remains in the recombined products. However, the minimum homology requirement in single-strand annealing is 29 bp (43) whereas MHMR uses 2–7 bp during nonhomologous integration (20). Several models of MHMR that mimic the mechanism of single-strand annealing have been proposed that involve 5′ to 3′ resection of the DSB and annealing of microhomology sequences, followed by gap-filling and ligation (5, 40).

MHMR is used preferentially in NHEJ-deficient cells and has been proposed as a backup pathway of NHEJ (44). Nonetheless, MHMR is a mutagenic pathway that generates large deletions and other genomic rearrangements, leading to human cancers and genetic diseases (1317). Based on our data, it is plausible that part of the single-strand annealing apparatus interacts with other genetic factors that are involved in modulating the homology requirement in MHMR and allowing illegitimate recombination between a few base pairs of homology. Investigation of these genetic factors will be important in understanding the molecular mechanism of the MHMR pathway.

Supplementary Material

Acknowledgments

This research was supported in part by project 1 to RHS of NIH grant 1 U19 AI 67769-01 to William McBride and a fellowship of the UCLA Chemistry-Biology Interface Training Program with USPHS National Research Service Award GM08496 and a research fellowship of the UC Toxic Substances Research and Teaching Program, both to CYC.

Footnotes

SUPPLEMENTARY INFORMATION

Tables S1–S5. The genomic location, locus and gene of the target sites in the rad51, rad52, rad1, rad10 and rad2 mutants. http://dx.doi.org/10.1667/RR1675.1.S1

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