And Tel1-Dependent Single-Strand DNA Formation at DNA Break Promotes Microhomology-Mediated End Joining

Saccharomyces cerevisiae Sae2- and Tel1-Dependent Single-Strand DNA Formation at DNA Break Promotes Microhomology-Mediated End Joining

Kihoon Lee and Sang Eun Lee1 Department of Molecular Medicine and Institute of Biotechnology University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245 Manuscript received May 24, 2007 Accepted for publication May 25, 2007

ABSTRACT Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches ½5–20 nucleotides (nt) of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single- stranded DNA (ssDNA) via a few bases (,5 nt) or extensive direct repeat homologies (.20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 39 ﬂap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.

NA double-strand breaks (DSBs) are extremely 1997; Schar et al. 1997; Wilson et al. 1997; Boulton D deleterious to a cell, and if left unrepaired or mis- and Jackson 1998; Herrmann et al. 1998). Multiple repaired, cell death will ensue (Khanna and Jackson factors including POL4, FEN1, and artemis process ends 2001; Bassing and Alt 2004). All eukaryotes including to configure them for ligation (Wilson and Lieber the budding yeast Saccharomyces cerevisiae evolved mul- 1999; Wu et al. 1999; Tseng and Tomkinson 2004). In tiple mechanisms to eliminate DSBs. Homologous re- yeast, Mre11/Rad50/Xrs2 functions in end joining by combination (HR) and non-homologous end joining aligning and tethering DNA ends for ligation (Chen et al. (NHEJ) are the two DSB repair mechanisms that are 2001), whereas in mammals such activities involve the characterized in some detail (Paques and Haber 1999). scid gene product, a catalytic subunit of DNA-dependent HR faithfully reconstitutes genetic information by copy- protein kinase (DNA-PK) (Cary et al. 1997). Most recently, ing from a template homologous to the sequence flank- Cernunnos/XLF, a likely homolog of yeast NEJ1, has ing the DSB (Sung et al. 2000). All three subclasses of been identified in a screen for mutations that cause sev- HR, gene conversion, break-induced replication (BIR), ere combined immunodeficiency in human patients and single-strand annealing (SSA), depend on functional (Ahnesorg et al. 2006; Buck et al. 2006). RAD52 epistasis group genes (Paques and Haber 1999; In addition to these two mechanisms, a third DSB re- Symington 2002). In contrast, NHEJ joins broken ends pair pathway that operates independently of Rad52 and together with a few base pair deletions and/or inser- Ku proteins has been described for some time. The pres- tions regardless of sequence homology (Critchlow ence of this mechanism is most evident when HR and/ and Jackson 1998; Daley et al. 2005b; Hefferin and or NHEJ are not available for repair, e.g., owing to muta- Tomkinson 2005). NHEJ requires the evolutionary con- tions in mouse Ku86 or deletion of yeast KU70 and RAD52 served KU70/KU80 and DNL4/LIF1 (Ligase 4 and Xrcc4 genes (Liang and Jasin 1996; Boulton and Jackson in mammals) proteins for end binding and ligation 1998; Yu and Gabriel 2003). Nevertheless, when a DNA (Rathmell and Chu 1994; Taccioli et al. 1994; break occurs with no complementary end sequence for Nussenzweig et al. 1996; Bogue et al. 1997; Gu et al. alignment or homologous template sequence for recombination, Rad52- and KU-independent microhomology- mediated end joining (MMEJ) becomes the primary 1Corresponding author: Department of Molecular Medicine and Institute repair choice (Ma et al. 2003). In Drosophila melanogaster, of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245. P-element-excised DNA DSBs are primarily eliminated E-mail: [email protected] by a DmRad51 (spn-A)-mediated synthesis-dependent

Genetics 176: 2003–2014 (August 2007) 2004 K. Lee and S. E. Lee strand annealing (SDSA) mechanism. However, in spn-A found in HeLa cell extracts that catalyze Ku-indepen- mutants repair proceeds through a nonconservative dent backup end joining (Wang et al. 2003). Recently, pathway involving the annealing of microhomologies we identified a role for the yeast Mre11 complex and within the 17-nt overhangs produced by P-element Rad1, a component of the structure-specific Rad1/ excision (McVey et al. 2004). End-to-end fusion of short Rad10 endonuclease, in repair of DNA DSBs with no telomeres in an Arabidopsis ku tert mutant occurs by complementary end sequences. These repair events MMEJ (Heacock et al. 2004). In mammals, a backup occurred primarily by MMEJ-mediated annealing of end-joining pathway kinetically slower than the Ku- recessed microhomologies on either side of the broken dependent pathway has been revealed by biochemical DNA ends (Ma et al. 2003). Partial reduction of MMEJ in fractionation of extracts from cells lacking DNA- dnl4D mutants also suggests a role for Dnl4 and Cdc9 in dependent protein kinase (Perrault et al. 2004). the sealing of DNA ends during MMEJ (Ma et al. 2003). Similarly, slower end-joining activity independent of Ku It is anticipated that SSA, NHEJ, and MMEJ all anneal proteins has been reported from studies in Xenopus DNA ends via recessed microhomology on opposite extracts and chicken DT-40 cells (Gottlich et al. 1998; sides of two broken DNA ends and remove 39 non- Takata et al. 1998; Labhart 1999). MMEJ is thus an homologous tails prior to gap filling and ligation as evolutionarily well-conserved mechanism to eliminate illustrated in Figure 1. Therefore, MMEJ may be mech- DSBs even when NHEJ and HR can in principle carry anistically similar to the Rad52-dependent SSA or Ku- out this function. dependent NHEJ mechanisms and likely shares some MMEJ is highly mutagenic and likely contributes to genetic components with these two processes. In sup- genome instability found in certain cancer cells. It port of this model, Rad1 and Mre11 participate in SSA, involves base pairing between microhomologies that whereas Dnl4 functions in NHEJ (Ma et al. 2003). We always leads to deletion of intervening sequence be- predict that additional NHEJ or SSA factors may par- tween the microhomologies (Ma et al. 2003; Daley and ticipate in MMEJ. To test this hypothesis, we surveyed Wilson 2005). Moreover, MMEJ may be responsible for the role of many factors known to function in either gross chromosomal rearrangements observed in yeast NHEJ or SSA in the repair by MMEJ of two breaks with mutants with a compromised DNA damage response no complementary end sequences. This study revealed a (Kolodner et al. 2002). Lack of NHEJ and/or HR also dozen genes involved in MMEJ and their catalytic func- leads to a dramatic increase in chromosome abnormal- tions during MMEJ reactions. The results also validate ities such as translocations and deletions in both yeast our model that MMEJ is mechanistically similar to SSA and mice (Davies et al. 1995; Pierce and Jasin 2001; Yu and Ku-dependent NHEJ. and Gabriel 2003). Remarkable similarity between junctional sequences of MMEJ repair and those from human disease-causing chromosomal translocations un- derscores the role of MMEJ in oncogenic chromosome MATERIALS AND METHODS u abriel rearrangements (Y and G 2003). In accordance Strains and plasmids: All strains are derivatives of SLY19, with this idea, repair of DNA breaks in human bladder which has the genotype hoD MATaTURA3THOcs hmlDTADE1 cancer cells occurs by an alternative mutagenic end- hmrDTADE1 ade1-100 leu2-3,112 lys5 trp1ThisG ura3-52 ade3T joining pathway that is mediated by microhomology GALTHO (Ma et al. 2003) (supplemental Table 1 at http:// (Bentley et al. 2004). Most pathological DSBs occur- www.genetics.org/supplemental). All single gene deletion mutants were constructed by using a PCR-derived KANMX ring in vivo may not have complementary overhangs for module flanked by short terminal sequences homologous to base pairing or homologous sister chromatids in the G1 the ends of each single gene open reading frame (Wach et al. phase (Lieber 1999). MMEJ may thus constitute a cri- 1994). Double mutant strains were constructed by first replac- tical means for cells to repair breaks in this phase of the ing the KANMX selectable marker from the single gene cell cycle. Indeed, MMEJ contributes almost equally with deletion using a DNA fragment containing the TRP1 marker and then deleting the second gene by one-step gene replace- Ku-dependent NHEJ to the repair of ionizing radiation- ment using the now available KANMX module again. Strain induced DNA damage (Ma et al. 2003). SLY19-mre11D was transformed with centromeric plasmids Despite its impact on genome integrity, little is known carrying either MRE11, mre11-3, mre11-H125N, or mre11-P162S, about the genetic requirements of MMEJ. In budding or an empty vector to test for the effect of MRE11 mutations on MMEJ. Similarly, SLY19-pol4D was transformed with plas- yeast, MMEJ is independent of Rad52 and Ku proteins ilson ieber a aley ilson mids pTW300, pTW301, or pTW305 (W and L (M et al. 2003; D and W 2005). In fact, Ku 1999) to investigate the role of Pol4 in MMEJ. may be inhibitory to non-Ku-mediated end joining that HO endonuclease induction: HO endonuclease was in- includes MMEJ (Boulton and Jackson 1998). Bio- duced according to the protocol described previously (Ma chemical fractionation of Ku-independent end-joining et al. 2003). Briefly, serial dilutions of cells grown to mid-log activity in Xenopus egg extracts uncovered roles for phase in YEP-glycerol at 30° were plated onto YEP-agar medium containing either galactose (YEP–GAL) or glucose (YEPD). DNA ligase III, Pole, Fen1, and an unidentified bidirec- The frequency of survival following an HO-induced DSB was tional exonuclease activity in the error prone end-joining calculated by dividing the number of colonies growing on reaction (Gottlich et al. 1998). DNA ligase III was also YEP–GAL by the number of colonies growing on YEPD. Factors in Microhomology-Mediated End Joining 2005

Figure 1.—A model of the biochemical steps that lead to MMEJ, NHEJ, and SSA. Following resection, MMEJ, NHEJ, and SSA all anneal microhomologies located at either side of the break, followed by end processing including 39 ﬂap removal, gap ﬁlling, and ligation of broken ends to complete repair.

Analysis of repair events: Colonies growing on YEP–GAL cells survive these breaks primarily by MMEJ (80%) or plates were replica plated onto synthetic complete (SC) me- less frequently by Ku-dependent NHEJ (20%), which dium lacking uracil, and mating type was determined by com- renders the HO recognition sites resistant to additional plementation using tester strains. Colonies displaying uracil a auxotrophy and mating-type a were selected for analysis of enzyme cleavage (M et al. 2003). repair events by amplifying the region spanning the repair SLY19 derivative strains deleted for EXO1, MSH2, junctions with PCR using a set of primers: pX (59-GTAAA MSH3, MSH6, NEJ1, POL4, RAD10, RAD27, RAD51, RAD59, CGGTGTCCTCTGTAAGG-39) and p2 (59-TCGAAAGATAAA SAE2, SGS1,orSRS2, all of which have roles in NHEJ, 9 CAACCTCC-3 ) and sequencing. SSA, or both (Paques and Haber 1999; Symington Chromatin immunoprecipitation: Chromatin immunopre- aley cipitation (ChIP) was carried out as described previously with 2002; D et al. 2005b), were induced for HO expres- minor modifications (Sugawara et al. 2003). Briefly, cultures sion by plating onto galactose-containing medium grown to 3 3 106 cells/ml in pre-induction medium (YEP– (YEP–GAL). Survival rates were determined from the glycerol) were induced for HO endonuclease by addition of number of colonies growing on galactose-containing 2% (final concentration) galactose, crosslinked by 1% form- plates, normalized by that on glucose-containing me- aldehyde (final volume), and sonicated to an average size of 0.5 kb. The sonicated extracts were incubated with anti-RPA dium (Figure 2B). To determine whether the repair of antibody (kindly provided by S. Brill) at 4° for 2 hr and then HO-induced breaks in SLY19 occurred by NHEJ or with protein G agarose bead for 1 hr. PCR condition and the Ku-independent MMEJ, yeast strains deleted for both primers used for ChIPs are described previously (Shim et al. KU70 and each NHEJ or SSA gene were tested for their 2005). survival by MMEJ only. Types of repair events that occur in the survivors were determined by PCR amplification and sequencing of the region spanning repair junctions RESULTS using a set of oligonucleotides that anneal to the regions Identification of additional MMEJ genes that also 59 to the 117-bp MATa cleavage site and 39 to the MATa function in NHEJ or SSA: Predicted similarity between HO recognition site (Figure 2C and supplemental Table MMEJ, SSA, and Ku-dependent NHEJ (Figure 1) sug- 2 at http://www.genetics.org/supplemental). Junctions gests that additional NHEJ or SSA factors besides the using .5 bp of microhomology were attributed to the Mre11 complex, Rad1, and Dnl4 may contribute to MMEJ product, while those using fewer than 5 bp of mi- MMEJ (Ma et al. 2003). Using an assay in S. cerevisiae that crohomology for base pairing were by Ku-dependent measures the repair of two simultaneous DSBs with no NHEJ (Ma et al. 2003). complementary end sequences, we surveyed the role of The approach described above identified four new almost every NHEJ or SSA factor in MMEJ. Expression of MMEJ genes, all of which have known roles in either a galactose-inducible HO gene inserted at the ADE3 NHEJ or SSA. Deletion of NEJ1, POL4, RAD10,orSRS2 locus simultaneously cleaves two inversely oriented HO from SLY19 or the KU70 deletion derivative of SLY19 recognition sites, a 117-bp sequence from MATa and the (SLY19-ku70D) reduced cell survival from HO breaks, MATa sequence, separated by 2.0 kb of DNA contain- revealing their role in MMEJ (Figure 2B). Analysis of ing the URA3 gene and Ya sequence (SLY19) (Figure repair junctions from survivors confirmed their involve- 2A). Lack of homologous templates (HML and HMR) ment in MMEJ, as the majority of junctions from strains effectively eliminates repair of the DSBs by HR. Thus, deleted for these genes retain features indicative of their 2006 K. Lee and S. E. Lee

Figure 2.—MMEJ frequency in various mutants. (A) Cleavage at two HO sites in the MAT locus of SLY19 separated by 2kbof URA3 sequence in opposite orien- tation generates noncomplemen- tary breaks that are preferentially joined by Ku- and Rad52- independent MMEJ. The location of primers for PCR amplification and sequencing are shown by ar- rows. (B) Survival frequency after induction of HO breaks of each mutant strain (shaded bars) and the corresponding KU70 deletion derivatives (solid bars). The frequency of survival after an HO- induced DSB was calculated by dividing the number of colonies growing on YEP–GAL by the number of colonies growing on YEPD. Each value represents the average from at least three independent experiments 6 standard deviation. (C) Junctional sequences observed in survivors of SLY19 and the mutant derivatives were used to calculate the frequency of repair events carried out by MMEJ or NHEJ. Typically, MMEJ involves .5 bp of imperfect microhomology, whereas NHEJ joins ends using ,5 nt of complementary base pairing. The region spanning junctional sequences was PCR amplified with a set of primers ½p2 and pX (A) that anneal to the proximal and distal regions of the HO cleavage sites and align with the original sequence of SLY19. For each mutant, between 24 and 96 survivors were sequenced (see supplemental Figure 2). formation by Ku-dependent NHEJ using fewer than 5 bp Provided that MMEJ and NHEJ occur primarily at of microhomology for base pairing, but junctions by different phases of the cell cycle, alteration of the cell MMEJ that use .5 bp of microhomology disappear cycle can account for the reduced MMEJ and a con- from the mutants (Figure 2C). It is possible, however, comitant increase in NHEJ among the newly identified that an assay analyzing repair events from unselected MMEJ mutants. This is unlikely, however, as the parallel total cell population may yield a different outcome. experiments with the nocodazole-arrested MMEJ mu- Interestingly, deletion of SAE2 increased cell survival tants at G2 prior to galactose addition revealed that the on YEP–GAL by 10-fold, although deletion of SAE2 MMEJ deficiency was identical to that without synchro- and KU70 reduced cell survival from the HO breaks to a nization (supplemental Figure 1 at http://www.genetics. level similar to that of rad10D ku70D, suggesting that org/supplemental). Flow cytometry also failed to detect increased survival of the sae2D mutant arose from in- substantial change in cell-cycle patterns among MMEJ creased KU-dependent NHEJ, but not from enhanced mutants (supplemental Figure 2). The results support MMEJ (Figure 2B). Survival rates and the sequence of our hypothesis that MMEJ, NHEJ, and SSA often rely on repair junctions indeed indicated that the deletion of the same gene products to catalyze similar reactions for SAE2 severely reduced MMEJ, which was compensated DSB repair. for by elevated Ku-dependent NHEJ (Figure 2C). De- Mre11 resects DNA ends to reveal and anneal letion of mismatch repair genes (MSH2, MSH3, MSH6, microhomology in MMEJ: Having established a role and EXO1), RAD52 epistasis group genes (RAD51 and for several genes and their gene products in MMEJ, we RAD59), FEN1,orSGS1 in either SLY19 or the SLY19- set out to determine how these proteins catalyze MMEJ yku70D strains did not significantly (Student’s t-test, P . reactions. Previously, we discovered that MMEJ depends 0.1) alter cell survival after DSB induction nor the type on Mre11 and Rad50 and likely Xrs2 as well (Ma et al. of repair events (mostly MMEJ) among survivors (Figure 2003) (Figure 3). Biochemical studies have shown that 2, B and C). Mre11 has double-strand exonuclease and single-strand Factors in Microhomology-Mediated End Joining 2007

bearing mutant mre11 constructs to assess the effect of the mre11 mutations on cell survival after induction of persistent HO cleavage by plating cells onto galactose- containing medium (Figure 3A). The results were compared to SLY19-mre11D transformed with either the wild-type MRE11 expression construct or an empty vector. We also sequenced 100 PCR-amplified repair junctions from the survivors to ensure the repair events were mediated by 5 bp or more of microhomology, typical of MMEJ products. We found that expression of the nuclease defective mre11-H125N or mre11-3 mutations rescued lethality of the mre11 null mutant upon DSB induction (Figure 3A). However, analysis of repair junctions from survivors revealed that almost all were repaired by Ku-dependent NHEJ rather than MMEJ (Figure 3A and supplemental Table 2 at http://www. genetics.org/supplemental). Expression of an all but null allele of mre11-P162S (Chamankhah et al. 2000) markedly reduced cell survival from HO breaks both by MMEJ and NHEJ. Our interpretation of these results is that the nuclease activity of Mre11 is critical for MMEJ. Overproduction of the 59 to 39 exonuclease Exo1 was previously shown to complement the resection defect of the mre11D mutant (Tsubouchi and Ogawa 2000; Moreau et al. 2001; Lee et al. 2002; Lewis et al. 2002). In addition, the 59 to 39 resection defect of the mre11D exo1D double mutant is more severe than that of the mre11D strain (Tsubouchi and Ogawa 2000). If Mre11 Figure 3.—The role of Mre11 nuclease activity in MMEJ. participates in MMEJ as a nuclease to create single- (A) SLY19-mre11D was transformed with yeast centromeric plas- stranded DNA (ssDNA) at DSBs, Exo1p may rescue the mids expressing MRE11, mre11-H125N, mre11-3, mre11-P162S, MMEJ deficiency of the mre11D strain. We overexpressed or an empty vector. Survival frequency and the relative contri- EXO1 in SLY19-mre11D and assayed the MMEJ defect of bution of MMEJ and NHEJ to survival after induction of HO this strain by measuring survival after HO cleavage and breaks were determined by analysis of junctional sequences observed in survivors. (B) Survival frequency of SLY19-exo1D, cataloging the repair events from sequencing the repair SLY19-exo1D mre11D, or SLY19-mre11D expressing additional junctions. As shown in Figure 3B, overproduction of Exo1 Exo1 from a GAL promoter. An empty vector containing but not the vector alone partially but consistently rescued the GAL promoter was used as a control. Each value repre- the decreased survival of SLY19-mre11D.Inaddition,an sents the average from at least three independent experi- mre11D exo1D double mutant showed a further reduction ments 6 standard deviation. in survival after HO expression rather than the mre11D mutant alone (Figure 3B). We thus propose that one endonuclease activities (Paull and Gellert 1998; function of the Mre11 complex in MMEJ is degrading Trujillo and Sung 2001) and a DNA hairpin endonu- 59 DNA ends to generate ssDNA to reveal microhomol- clease activity that can incise DNA at a single-strand/ ogy for annealing. This suggests that in the absence of duplex junction (Paull and Gellert 1999; Trujillo Mre11, other nuclease(s) such as Exo1 can provide less and Sung 2001). We thus reasoned that Mre11 func- efficient end resection for the limited MMEJ reactions. tions to resect DNA ends in the MMEJ reaction. Should Rad1/Rad10 and Srs2 are involved in MMEJ: Rad1 the Mre11 complex provide the nuclease activity in and Rad10 form a heterodimeric structure-specific en- MMEJ, we predicted that the mre11 mutations that inac- donuclease that removes 39 flaps during HR (Sugawara tivate the nuclease activity, such as the mre11-H125N et al. 1997). Rad1/Rad10 likely participates in MMEJ by (Moreau et al. 1999) and mre11-3 mutations (Bressan catalyzing 39 flap removal after annealing of microho- et al. 1998), will also impair MMEJ. The mre11-H125N mology between ssDNA (Ma et al. 2003). Consistent with mutant has been confirmed biochemically to be defi- their function as a complex, a yeast strain deleted for cient for nuclease activity but maintains the ability to both RAD1 and RAD10 showed identical MMEJ de- form a heterotrimeric complex with Rad50 and Xrs2 ficiency as the rad1D and rad10D single mutant strains (Moreau et al. 1999). (Figure 2, B and C). The mre11 gene deletion derivative of SLY19 (SLY19- Srs2 suppresses recombination by displacing Rad51 mre11D) was transformed with centromeric plasmids from ssDNA (Krejci et al. 2003; Veaute et al. 2003). Srs2 2008 K. Lee and S. E. Lee

TRF4 did not confer any detectable MMEJ deficiency. Potential involvement of multiple polymerases in MMEJ prompted us to consider redundancy among polymerases in the gap-filling step of MMEJ. We tested this idea by constructing yeast strains with two or more polymerase gene deletions and assessed their MMEJ deficiency by analyzing repair events following HO cleavage. None of double mutant strains showed additional MMEJ deficiency beyond that of each single gene deletion mutant (Figure 5B). However, deletion of POL32, RAD30, and REV3 together almost completely wiped off any residual MMEJ among survivors (Figure 5B). The results thus reveal redundancy among these polymerases in MMEJ repair. Pol4 is not a processive polymerase (Bebenek et al. 2005). It mostly fills small gaps without synthesizing long aley Figure 4.—Lack of Rad51 rescues the MMEJ deficiency of stretches of nucleotides (D et al. 2005a). Lack of srs2D. Survival frequency following induction of HO breaks of processivity led us to consider the possibility that Pol4 srs2D, rad51D, and srs2D rad51d strains (shaded bars) and the plays a role in MMEJ separate from its DNA polymeri- corresponding KU70 deletion derivatives (solid bars) were de- zation activity. Previously, the BRCT domain of Pol4 was termined as described in Figure 2. The average from at least three independent experiments 6 standard deviation is shown to interact with the Dnl4/Lif1 complex and to shown. couple the gap filling and ligation reactions during Ku- dependent NHEJ (Tseng and Tomkinson 2002, 2004). Consistent with the partial dependency of MMEJ on Dnl4, also performs prorecombination functions during alle- we reasoned that Pol4 may facilitate the ligation step of lic recombination and SSA (Sugawara et al. 2000; Ira MMEJ by recruiting Dnl4/Lif1 to the repair site. To test et al. 2003). In SSA, Srs2 may stabilize homologous joints this hypothesis, a yeast centromeric plasmid expressing and promote the removal of 39 flap DNA with the Rad1/ either the BRCT domain-truncated pol4 (pol4Dbr)mu- Rad10 complex by relieving superhelical stress from tation or the polymerase-dead pol4-D367E mutation unwinding donor duplex DNA (Paques and Haber (Wilson and Lieber 1999) was introduced into the 1997). Alternatively, Srs2 can promote MMEJ by displac- POL4-deleted SLY19 derivative. Expression of pol4Dbr ing Rad51 and thus inhibiting recombination of resected failed to rescue MMEJ repair in pol4D, suggesting that DNA ends. To distinguish between these two possibili- the BRCT domain is required for repair by MMEJ (Fig- ties, we deleted RAD51 in an srs2D strain and investi- ure 5C). More importantly, expression of the polymerase- gated its ability to undergo MMEJ repair of an HO defective pol4-D367E mutation partially rescued the break. We found that deletion of RAD51 fully rescued lethality of an HO break in the pol4D mutant by elevated the MMEJ repair defect in srs2D (Figure 4). This result Ku-dependent NHEJ but failed to recover the MMEJ suggests that Srs2 is involved in MMEJ as an antirecom- deficiency. Analysis of repair events among survivors of binase by displacing Rad51 from ssDNA. HO breaks confirmed that most survivors repaired DSBs Polymerase activity of Pol4 is essential for gap fill-in by NHEJ via 2 bp of microhomology, deleting 5 nt at the after annealing between microhomology: MMEJ likely junctions without DNA synthesis (see supplemental Table depends on DNA polymerase(s) to fill gaps of various 2athttp://www.genetics.org/supplemental). These re- sizes ranging from a few to several hundred nucleotides sults support the hypothesis that Pol4 is required for gap following 59 nucleolytic degradation to expose micro- filling in MMEJ. homologies within the regions flanking a DSB (Figure Sae2 and Tel1 promote MMEJ but inhibit NHEJ: 1). In addition to Pol4, we tested two other nonessential Sae2, along with the Mre11 complex, is required for polymerases (Rev3 and Rad30), Pol32 (a subunit of cleavage of Spo11 bound to DNA ends to initiate meio- Pold), and poly (A) RNA polymerase Trf4 (Holbeck tic recombination (McKee and Kleckner 1997; Prinz and Strathern 1997; McDonald et al. 1997; Gerik et al. 1997). Sae2 also contributes to SSA between direct et al. 1998; Wang et al. 2000; Zhao et al. 2004, 2006), for repeat sequences by promoting the nuclease activity of their role in MMEJ by determining survival of single- the Mre11 complex and tethering broken DNA ends in deletion mutant strains following HO expression (Fig- mitotic cells (Clerici et al. 2005). Interestingly, deletion ure 5A). We found that deletion of POL4, POL32, of SAE2 or expression of a nuclease defective mre11 allele RAD30,orREV3 all led to a 3- to 10-fold reduction in (mre11-H125N) results in a very similar phenotype— survival from the HO break because of a combined de- an increase in Ku-dependent NHEJ but reduced MMEJ ficiency in NHEJ and MMEJ (pol4D) or the MMEJ defect (Figures 2, B and C and 3A). We questioned whether Sae2 only (pol32D, rad30D, and rev3D). Yeast strains lacking contributes to MMEJ by promoting Mre11-dependent Factors in Microhomology-Mediated End Joining 2009

Figure 5.—MMEJ requires multiple polymerases including two translesion bypass polymerases. Survival frequency (A) and the contribution of MMEJ and NHEJ to repair (B) among survivors after induction of HO breaks of strains lacking one or more nonessential DNA polymerases or KU70 were determined by analysis of junctional sequences observed in survivors. (C) Plasmids expressing POL4 or its mutant derivatives were introduced into SLY19-pol4D, and the frequency of MMEJ and NHEJ following induction of HO breaks was determined as in A. Each value represents the average from at least three independent experiments 6 standard deviation.

nucleolytic degradation but inhibiting Ku-dependent We also assessed the role of Sae2 and Tel1 in NHEJ by NHEJ. If this is the case, we predict that overexpression examining the effect of a sae2 or tel1 deletion in SLY1 of EXO1 may partially rescue the MMEJ deficiency of and SLY18 strains (Figure 6, C and D). Repair of a sin- sae2D or sae2D mre11D and simultaneously reduce gle HO break in these two strains occurs primarily by NHEJ. Increased expression of EXO1 had no impact Ku-dependent NHEJ. We found that deletion of SAE2 on MMEJ or NHEJ in sae2D but significantly increased or TEL1 improved cell survival up to eightfold by survival of sae2D mre11D mutants upon HO induction Ku-dependent NHEJ (Figure 6, C and D). The results with a modest increase in MMEJ repair (Figure 6B). The suggest that suppression of NHEJ by Sae2 is not unique results thus support the model that Sae2-dependent 59 to SLY19 and is independent of the MMEJ pathway. resection plays a regulatory role in modulating MMEJ Increase in NHEJ efficiency at the expense of de- and NHEJ. creased MMEJ from these mutants after persistent HO Sae2 is a phosphoprotein whose phosphorylation de- cleavage may be explained by the reduced 59 resection pends on the Mec1/Tel1 kinases (yeast ATR and ATM (Clerici et al. 2005; Mantiero et al. 2007), as the extent homologs, respectively) and DNA damage (Baroni et al. of nucleolytic degradation may be an impediment for 2004). To test the effect of phosphorylation of Sae2 on NHEJ (Ira et al. 2004). We thus determined the rate of MMEJ, we deleted TEL1 or MEC1/SML1 from SLY19 resection at the DSB from sae2D or tel1D strain by mea- and measured their effect on MMEJ efficiency following suring the level of replication protein A (RPA) binding, two HO breaks. We discovered that there was a fivefold a trimeric single-strand binding protein, to ssDNA increase in NHEJ compared to wild type, but that rare formed at or near the DSB with ChIP assay followed by MMEJ repair of two simultaneous HO breaks was re- qPCR using primer sets annealed to multiple regions duced in the TEL1-deleted strain (Figure 6A). These at the MAT locus (Shim et al. 2005). We found that data suggest that Sae2 and Tel1 constitute an important depletion of SAE2 or TEL1 reduced the replication control for antagonistic modulation of MMEJ and protein A (RPA) binding to the DSB and thus decreased NHEJ. the formation of ssDNA (Figure 6, E–H). 2010 K. Lee and S. E. Lee

Figure 6.—Roles of Sae2, Mec1, and Tel1 in MMEJ and NHEJ. Contribution of MMEJ and NHEJ to repair among survivors after induction of HO breaks of sae2D, tel1D, and mec1Dsml1D strains in A or sae2D and sae2D mre11D expressing additional Exo1 in B were determinedasdescribed inFigure2.SurvivalfrequencyfollowinginductionofHObreaksinsae2Dor tel1DderivativesofSLY1inCand SLY18in D,which carry oneor twoHO cleavagesites indirect repeatorientation inthe MAT locus, weredetermined byplatingcells on YEP–GAL normalized by the plating efficiency on YEPD. Each value represents the average from at least three independent experiments 6 standard deviation. HO cleavage sites in the MAT locus in SLY1 or SLY18 are shown schematically above each graph. In E–H ChIP assays were used to assess the levels of RPA in sae2D or tel1D at the DSB using an anti-RPA antibody. Chromatin was isolated at the indicated time after galactose addition, crosslinked with formaldehyde, and fragmented by sonication. After immunoprecipitation andreverse crosslinking, purified DNA was analyzed by qPCR using three sets of primers that anneal 0.2 kb in F,1 kb in G, and5 kb in H to the DSB, as well as primers specific for the PRE1 gene situated on chromosome V as a control. PCR signals from each primer set at different durations of HO expression were quantified and plotted as a graph. IP represents the ratio of the RPA PCR signal before and after HO induction, normalized by the PCR signal of the PRE1 control. The positions of HO cut site and the location of primers are shown in E. Each point is the average of two separate experiments.

also found that nucleolytic degradation suppresses NHEJ, DISCUSSION whereas it is essential for MMEJ. We propose that MMEJ To assess the mechanistic overlap between MMEJ, is achieved by a series of distinct but mechanistically NHEJ, and SSA, we surveyed the majority of known similar biochemical reactions that also occur in NHEJ NHEJ or SSA factors for their roles in MMEJ repair of and SSA. two DSBs lacking complementary base overhangs. The Role of ssDNA formation at DNA DSBs in MMEJ analysis newly identified genes responsible for MMEJ. and NHEJ: MMEJ requires the Mre11 complex to repair Those include NEJ1, POL4, POL32, RAD10, RAD30, REV3, two nearby DSBs without complementary end sequen- SRS2, SAE2, and TEL1. In addition, this study provided ces (Ma et al. 2003). By examining the effect of mre11 some insight into their specific functions in MMEJ. We mutations, which impair various enzymatic functions of Factors in Microhomology-Mediated End Joining 2011 the wild-type protein, we discovered that the nuclease 2004), we propose that this complex catalyzes 39 flap activity of the Mre11 complex is critical for MMEJ. The removal in MMEJ. importance of Mre11’s nuclease activity in MMEJ is We demonstrated that deletion of SRS2 displayed re- further supported by the observed partial complemen- duced MMEJ frequency, which can be reversed by simul- tation of MMEJ deficiency in mre11D mutant cells by taneous deletion of RAD51. We imagine that ssDNA overproduction of yet another nuclease Exo1p. Dele- formed by Mre11-dependent 59 resection of DSBs will tion of SAE2, a key component of meiotic DSB process- attract binding of single-strand binding proteins such as ing and mitotic SSA, or TEL1 also led to reduced MMEJ RPA or Rad51. Given its function as a strand-annealing repair of two simultaneous DSBs. In addition, reduced protein between ssDNA and duplex DNA molecules MMEJ in the mre11D sae2D mutant is partially rescued by (Sung 1994), Rad51 binding to ssDNA may interfere expressing EXO1 in excess. Together, these data provide with annealing between single-stranded DSB ends dur- compelling evidence that end processing by the Mre11 ing SSA or MMEJ. The inhibitory effect of Rad51 in SSA complex and Sae2 is a critical part of the MMEJ reaction. is supported by the finding that deletion of RAD51 We therefore reasoned that 59 resection activity should significantly improves the efficiency of SSA (Ira and uncover microhomologies at either side of the DSB for Haber 2002). Alternatively, Rad51 may interfere with annealing during the MMEJ reaction. binding of yet another single-strand binding protein Although it is an important feature of the MMEJ that catalyzes annealing of ssDNA. Genetic and biochem- repair reaction, 59 resection actually suppresses NHEJ, ical evidence strongly suggests that Srs2 is an antire- as the nuclease defective mre11 mutation or a SAE2 dele- combinase, displacing Rad51 from ss- or double-stranded tion greatly stimulated Ku-dependent NHEJ. Previously, DNA (dsDNA) molecules (Krejci et al. 2003; Veaute unprocessed DNA ends were identified as a preferred et al. 2003). We propose that Rad51 displacement from substrate for NHEJ (Frank-Vaillant and Marcand ssDNA is necessary for efficient MMEJ, and Srs2 achieves 2002). NHEJ is most active in the G1 phase of the cell this through its Rad51 displacement activity. cycle when the nucleolytic processing of DNA ends is Multiple polymerases fill in gaps during MMEJ: Ana- less active (Ferguson et al. 2000; Aylon et al. 2004; Ira lysis of MMEJ repair events suggests that cells frequently et al. 2004). Together, these results suggest that nucle- use microhomologies, with one only a few bp away but olytic end processing affects both NHEJ and MMEJ but the other at 60 to 300 bp away from break. Provided that in opposite ways. It promotes MMEJ but limits NHEJ 59 resection exposing microhomology proceeds sym- reactions. The inhibitory effect of 59 resection on NHEJ metrically at either side of the break, MMEJ needs con- suggests that yKu may be inhibitory to the MMEJ pro- siderable DNA synthesis to fill these gaps for efficient cess. The absence of Ku accelerates nucleolytic degra- MMEJ. Interestingly, we found that at least three DNA dation (Liang and Jasin 1996; Lee et al. 1998) and can polymerases and one accessory factor of a major repli- stimulate the MMEJ reaction. The results also explain cative polymerase are needed to accomplish efficient why two DSBs with no complementary end sequences MMEJ. Why does MMEJ require multiple polymerases? are preferentially repaired by MMEJ rather than by One reason may be that each polymerase contributes to NHEJ (Ma et al. 2003). The lack of complementary bases MMEJ at different cell-cycle phases, locations, or se- prevents DSB ends from annealing, making them more quences. Indeed, Rad30 and Rev3 expression fluctuates vulnerable to nuclease activity. during the cell cycle (Saccharomyces Genome Database, The Mre11 complex and Sae2 are modified by the http://www.yeastgenome.org), whereas expression of Mec1 or Tel1 kinases upon DNA damage (Usui et al. Pol4 is constant throughout all phases. Alternatively, 2001; Baroni et al. 2004). Loss of Tel1 (and to a lesser the requirement for multiple polymerases may reflect extent Mec1) increased NHEJ but reduced MMEJ. Inter- that MMEJ is achieved by the ‘‘two polymerase two step estingly, Sae2 phosphorylation was shown to be critical model’’ originally proposed to account for multiple for processing and displacing Mre11 from DSB ends polymerase involvement in translesion synthesis (Wang (Clerici et al. 2005, 2006). Considering the role of nu- 2001). According to this model, one or more poly- cleolytic degradation in MMEJ and NHEJ, it seems logi- merases initiate gap synthesis, followed by extension cal to propose that phosphorylation of Sae2 modulates synthesis catalyzed by yet another polymerase(s). Pol4 is MMEJ by promoting DSB-induced DNA end resection. well suited to initiate 39-end synthesis from mismatches Role of Srs2 and Rad1/Rad10 in MMEJ: Following 59 at annealed microhomologies because of its relaxed degradation of DSB ends to generate 39 ssDNA, proper template dependency (Pardo et al. 2006). However, the microhomologies anneal, and the 39 flaps are removed low processivity of Pol4 prevents it from long gap fill-in for gap fill-in synthesis and ligation to complete MMEJ. synthesis. Indeed, recombinant Pol4 demonstrated very We showed that both Rad1 and Rad10 are needed for poor processivity in primer extension assays with ,25% MMEJ but dispensable for NHEJ. As Rad1 and Rad10 of a 5-nt gap filled in (Bebenek et al. 2005). Pold, Rad30, form an endonuclease complex that removes 39 flaps Rev3, or yet another polymerase(s) may contribute during gene conversion or SSA (Fishman-Lobell and to MMEJ by extending synthesis beyond the initial Haber 1992; Davies et al. 1995; Ferreira and Cooper synthesis by Pol4. As deletion of Pol4 did not completely 2012 K. Lee and S. E. Lee eliminate MMEJ, we also propose that another poly- Aylon, Y., B. Liefshitz and M. 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