Saccharomyces cerevisiae Sae2- and Tel1-Dependent Single ......synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes,

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.076539

Saccharomyces cerevisiae Sae2- and Tel1-Dependent Single-StrandDNA 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, 2007Accepted 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 endjoining (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). Althoughthe genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterizedin 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 MMEJrequires the nuclease activity of Mre11/Rad50/Xrs2, 39 flap removal by Rad1/Rad10, Nej1, and DNAsynthesis 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 inhibitNHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support therole of Sae2 and Tel1 in MMEJ and genome integrity.

DNA double-strand breaks (DSBs) are extremelydeleterious to a cell, and if left unrepaired or mis-

repaired, cell death will ensue (Khanna and Jackson

2001; Bassing and Alt 2004). All eukaryotes includingthe budding yeast Saccharomyces cerevisiae evolved mul-tiple mechanisms to eliminate DSBs. Homologous re-combination (HR) and non-homologous end joining(NHEJ) are the two DSB repair mechanisms that arecharacterized in some detail (Paques and Haber 1999).HR faithfully reconstitutes genetic information by copy-ing from a template homologous to the sequence flank-ing the DSB (Sung et al. 2000). All three subclasses ofHR, gene conversion, break-induced replication (BIR),and single-strand annealing (SSA), depend on functionalRAD52 epistasis group genes (Paques and Haber 1999;Symington 2002). In contrast, NHEJ joins broken endstogether with a few base pair deletions and/or inser-tions regardless of sequence homology (Critchlow

and Jackson 1998; Daley et al. 2005b; Hefferin andTomkinson 2005). NHEJ requires the evolutionary con-served KU70/KU80 and DNL4/LIF1 (Ligase 4 and Xrcc4in mammals) proteins for end binding and ligation(Rathmell and Chu 1994; Taccioli et al. 1994;Nussenzweig et al. 1996; Bogue et al. 1997; Gu et al.

1997; Schar et al. 1997; Wilson et al. 1997; Boulton

and Jackson 1998; Herrmann et al. 1998). Multiplefactors including POL4, FEN1, and artemis process endsto configure them for ligation (Wilson and Lieber

1999; Wu et al. 1999; Tseng and Tomkinson 2004). Inyeast, Mre11/Rad50/Xrs2 functions in end joining byaligning and tethering DNA ends for ligation (Chen et al.2001), whereas in mammals such activities involve thescid gene product, a catalytic subunit of DNA-dependentprotein kinase (DNA-PK) (Cary et al. 1997). Most recently,Cernunnos/XLF, a likely homolog of yeast NEJ1, hasbeen identified in a screen for mutations that cause sev-ere combined immunodeficiency in human patients(Ahnesorg et al. 2006; Buck et al. 2006).

In addition to these two mechanisms, a third DSB re-pair pathway that operates independently of Rad52 andKu proteins has been described for some time. The pres-ence of this mechanism is most evident when HR and/or NHEJ are not available for repair, e.g., owing to muta-tions in mouse Ku86 or deletion of yeast KU70 and RAD52genes (Liang and Jasin 1996; Boulton and Jackson

1998; Yu and Gabriel 2003). Nevertheless, when a DNAbreak occurs with no complementary end sequence foralignment or homologous template sequence for recom-bination, Rad52- and KU-independent microhomology-mediated end joining (MMEJ) becomes the primaryrepair choice (Ma et al. 2003). In Drosophila melanogaster,P-element-excised DNA DSBs are primarily eliminatedby a DmRad51 (spn-A)-mediated synthesis-dependent

1Corresponding author: Department of Molecular Medicine and Instituteof Biotechnology, University of Texas Health Science Center at SanAntonio, 15355 Lambda Drive, San Antonio, TX 78245.E-mail: [email protected]

Genetics 176: 2003–2014 (August 2007)

strand annealing (SDSA) mechanism. However, in spn-Amutants repair proceeds through a nonconservativepathway involving the annealing of microhomologieswithin the 17-nt overhangs produced by P-elementexcision (McVey et al. 2004). End-to-end fusion of shorttelomeres in an Arabidopsis ku tert mutant occurs byMMEJ (Heacock et al. 2004). In mammals, a backupend-joining pathway kinetically slower than the Ku-dependent pathway has been revealed by biochemicalfractionation of extracts from cells lacking DNA-dependent protein kinase (Perrault et al. 2004).Similarly, slower end-joining activity independent of Kuproteins has been reported from studies in Xenopusextracts and chicken DT-40 cells (Gottlich et al. 1998;Takata et al. 1998; Labhart 1999). MMEJ is thus anevolutionarily well-conserved mechanism to eliminateDSBs even when NHEJ and HR can in principle carryout this function.

MMEJ is highly mutagenic and likely contributes togenome instability found in certain cancer cells. Itinvolves base pairing between microhomologies thatalways leads to deletion of intervening sequence be-tween the microhomologies (Ma et al. 2003; Daley andWilson 2005). Moreover, MMEJ may be responsible forgross chromosomal rearrangements observed in yeastmutants with a compromised DNA damage response(Kolodner et al. 2002). Lack of NHEJ and/or HR alsoleads to a dramatic increase in chromosome abnormal-ities such as translocations and deletions in both yeastand mice (Davies et al. 1995; Pierce and Jasin 2001; Yu

and Gabriel 2003). Remarkable similarity betweenjunctional sequences of MMEJ repair and those fromhuman disease-causing chromosomal translocations un-derscores the role of MMEJ in oncogenic chromosomerearrangements (Yu and Gabriel 2003). In accordancewith this idea, repair of DNA breaks in human bladdercancer cells occurs by an alternative mutagenic end-joining pathway that is mediated by microhomology(Bentley et al. 2004). Most pathological DSBs occur-ring in vivo may not have complementary overhangs forbase pairing or homologous sister chromatids in the G1

phase (Lieber 1999). MMEJ may thus constitute a cri-tical means for cells to repair breaks in this phase of thecell cycle. Indeed, MMEJ contributes almost equally withKu-dependent NHEJ to the repair of ionizing radiation-induced DNA damage (Ma et al. 2003).

Despite its impact on genome integrity, little is knownabout the genetic requirements of MMEJ. In buddingyeast, MMEJ is independent of Rad52 and Ku proteins(Ma et al. 2003; Daley and Wilson 2005). In fact, Kumay be inhibitory to non-Ku-mediated end joining thatincludes MMEJ (Boulton and Jackson 1998). Bio-chemical fractionation of Ku-independent end-joiningactivity in Xenopus egg extracts uncovered roles forDNA ligase III, Pole, Fen1, and an unidentified bidirec-tional exonuclease activity in the error prone end-joiningreaction (Gottlich et al. 1998). DNA ligase III was also

found in HeLa cell extracts that catalyze Ku-indepen-dent backup end joining (Wang et al. 2003). Recently,we identified a role for the yeast Mre11 complex andRad1, a component of the structure-specific Rad1/Rad10 endonuclease, in repair of DNA DSBs with nocomplementary end sequences. These repair eventsoccurred primarily by MMEJ-mediated annealing ofrecessed microhomologies on either side of the brokenDNA ends (Ma et al. 2003). Partial reduction of MMEJ indnl4D mutants also suggests a role for Dnl4 and Cdc9 inthe sealing of DNA ends during MMEJ (Ma et al. 2003).

It is anticipated that SSA, NHEJ, and MMEJ all annealDNA ends via recessed microhomology on oppositesides of two broken DNA ends and remove 39 non-homologous tails prior to gap filling and ligation asillustrated in Figure 1. Therefore, MMEJ may be mech-anistically similar to the Rad52-dependent SSA or Ku-dependent NHEJ mechanisms and likely shares somegenetic components with these two processes. In sup-port of this model, Rad1 and Mre11 participate in SSA,whereas Dnl4 functions in NHEJ (Ma et al. 2003). Wepredict that additional NHEJ or SSA factors may par-ticipate in MMEJ. To test this hypothesis, we surveyedthe role of many factors known to function in eitherNHEJ or SSA in the repair by MMEJ of two breaks withno complementary end sequences. This study revealed adozen genes involved in MMEJ and their catalytic func-tions during MMEJ reactions. The results also validateour model that MMEJ is mechanistically similar to SSAand Ku-dependent NHEJ.

MATERIALS AND METHODS

Strains and plasmids: All strains are derivatives of SLY19,which has the genotype hoD MATaTURA3THOcs hmlDTADE1hmrDTADE1 ade1-100 leu2-3,112 lys5 trp1ThisG ura3-52 ade3TGALTHO (Ma et al. 2003) (supplemental Table 1 at http://www.genetics.org/supplemental). All single gene deletionmutants were constructed by using a PCR-derived KANMXmodule flanked by short terminal sequences homologous tothe ends of each single gene open reading frame (Wach et al.1994). Double mutant strains were constructed by first replac-ing the KANMX selectable marker from the single genedeletion using a DNA fragment containing the TRP1 markerand then deleting the second gene by one-step gene replace-ment using the now available KANMX module again. StrainSLY19-mre11D was transformed with centromeric plasmidscarrying either MRE11, mre11-3, mre11-H125N, or mre11-P162S,or an empty vector to test for the effect of MRE11 mutationson MMEJ. Similarly, SLY19-pol4D was transformed with plas-mids pTW300, pTW301, or pTW305 (Wilson and Lieber

1999) to investigate the role of Pol4 in MMEJ.HO endonuclease induction: HO endonuclease was in-

duced according to the protocol described previously (Ma

et al. 2003). Briefly, serial dilutions of cells grown to mid-logphase in YEP-glycerol at 30� were plated onto YEP-agar me-dium containing either galactose (YEP–GAL) or glucose (YEPD).The frequency of survival following an HO-induced DSB wascalculated by dividing the number of colonies growing onYEP–GAL by the number of colonies growing on YEPD.

2004 K. Lee and S. E. Lee

Analysis of repair events: Colonies growing on YEP–GALplates were replica plated onto synthetic complete (SC) me-dium lacking uracil, and mating type was determined by com-plementation using tester strains. Colonies displaying uracilauxotrophy and mating-type a were selected for analysis ofrepair events by amplifying the region spanning the repairjunctions with PCR using a set of primers: pX (59-GTAAACGGTGTCCTCTGTAAGG-39) and p2 (59-TCGAAAGATAAACAACCTCC-39) and sequencing.

Chromatin immunoprecipitation: Chromatin immunopre-cipitation (ChIP) was carried out as described previously withminor modifications (Sugawara et al. 2003). Briefly, culturesgrown to 3 3 106 cells/ml in pre-induction medium (YEP–glycerol) were induced for HO endonuclease by addition of2% (final concentration) galactose, crosslinked by 1% form-aldehyde (final volume), and sonicated to an average size of0.5 kb. The sonicated extracts were incubated with anti-RPAantibody (kindly provided by S. Brill) at 4� for 2 hr and thenwith protein G agarose bead for 1 hr. PCR condition and theprimers used for ChIPs are described previously (Shim et al.2005).

RESULTS

Identification of additional MMEJ genes that alsofunction in NHEJ or SSA: Predicted similarity betweenMMEJ, SSA, and Ku-dependent NHEJ (Figure 1) sug-gests that additional NHEJ or SSA factors besides theMre11 complex, Rad1, and Dnl4 may contribute toMMEJ (Ma et al. 2003). Using an assay in S. cerevisiae thatmeasures the repair of two simultaneous DSBs with nocomplementary end sequences, we surveyed the role ofalmost every NHEJ or SSA factor in MMEJ. Expression ofa galactose-inducible HO gene inserted at the ADE3locus simultaneously cleaves two inversely oriented HOrecognition sites, a 117-bp sequence from MATa and theMATa sequence, separated by �2.0 kb of DNA contain-ing the URA3 gene and Ya sequence (SLY19) (Figure2A). Lack of homologous templates (HML and HMR)effectively eliminates repair of the DSBs by HR. Thus,

cells survive these breaks primarily by MMEJ (�80%) orless frequently by Ku-dependent NHEJ (�20%), whichrenders the HO recognition sites resistant to additionalenzyme cleavage (Ma et al. 2003).

SLY19 derivative strains deleted for EXO1, MSH2,MSH3, MSH6, NEJ1, POL4, RAD10, RAD27, RAD51, RAD59,SAE2, SGS1, or SRS2, all of which have roles in NHEJ,SSA, or both (Paques and Haber 1999; Symington

2002; Daley et al. 2005b), were induced for HO expres-sion by plating onto galactose-containing medium(YEP–GAL). Survival rates were determined from thenumber of colonies growing on galactose-containingplates, normalized by that on glucose-containing me-dium (Figure 2B). To determine whether the repair ofHO-induced breaks in SLY19 occurred by NHEJ orKu-independent MMEJ, yeast strains deleted for bothKU70 and each NHEJ or SSA gene were tested for theirsurvival by MMEJ only. Types of repair events that occurin the survivors were determined by PCR amplificationand sequencing of the region spanning repair junctionsusing a set of oligonucleotides that anneal to the regions59 to the 117-bp MATa cleavage site and 39 to the MATa

HO recognition site (Figure 2C and supplemental Table2 at http://www.genetics.org/supplemental). Junctionsusing .5 bp of microhomology were attributed to theMMEJ product, while those using fewer than 5 bp of mi-crohomology for base pairing were by Ku-dependentNHEJ (Ma et al. 2003).

The approach described above identified four newMMEJ genes, all of which have known roles in eitherNHEJ or SSA. Deletion of NEJ1, POL4, RAD10, or SRS2from SLY19 or the KU70 deletion derivative of SLY19(SLY19-ku70D) reduced cell survival from HO breaks,revealing their role in MMEJ (Figure 2B). Analysis ofrepair junctions from survivors confirmed their involve-ment in MMEJ, as the majority of junctions from strainsdeleted for these genes retain features indicative of their

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

Factors in Microhomology-Mediated End Joining 2005

formation by Ku-dependent NHEJ using fewer than 5 bpof microhomology for base pairing, but junctions byMMEJ that use .5 bp of microhomology disappearfrom the mutants (Figure 2C). It is possible, however,that an assay analyzing repair events from unselectedtotal cell population may yield a different outcome.Interestingly, deletion of SAE2 increased cell survivalon YEP–GAL by �10-fold, although deletion of SAE2and KU70 reduced cell survival from the HO breaks to alevel similar to that of rad10D ku70D, suggesting thatincreased survival of the sae2D mutant arose from in-creased KU-dependent NHEJ, but not from enhancedMMEJ (Figure 2B). Survival rates and the sequence ofrepair junctions indeed indicated that the deletion ofSAE2 severely reduced MMEJ, which was compensatedfor by elevated Ku-dependent NHEJ (Figure 2C). De-letion of mismatch repair genes (MSH2, MSH3, MSH6,and EXO1), RAD52 epistasis group genes (RAD51 andRAD59), FEN1, or SGS1 in either SLY19 or the SLY19-yku70D strains did not significantly (Student’s t-test, P .

0.1) alter cell survival after DSB induction nor the typeof repair events (mostly MMEJ) among survivors (Figure2, B and C).

Provided that MMEJ and NHEJ occur primarily atdifferent phases of the cell cycle, alteration of the cellcycle can account for the reduced MMEJ and a con-comitant increase in NHEJ among the newly identifiedMMEJ mutants. This is unlikely, however, as the parallelexperiments with the nocodazole-arrested MMEJ mu-tants at G2 prior to galactose addition revealed that theMMEJ deficiency was identical to that without synchro-nization (supplemental Figure 1 at http://www.genetics.org/supplemental). Flow cytometry also failed to detectsubstantial change in cell-cycle patterns among MMEJmutants (supplemental Figure 2). The results supportour hypothesis that MMEJ, NHEJ, and SSA often rely onthe same gene products to catalyze similar reactions forDSB repair.

Mre11 resects DNA ends to reveal and annealmicrohomology in MMEJ: Having established a rolefor several genes and their gene products in MMEJ, weset out to determine how these proteins catalyze MMEJreactions. Previously, we discovered that MMEJ dependson Mre11 and Rad50 and likely Xrs2 as well (Ma et al.2003) (Figure 3). Biochemical studies have shown thatMre11 has double-strand exonuclease and single-strand

Figure 2.—MMEJ frequency invarious mutants. (A) Cleavage attwo HO sites in the MAT locus ofSLY19 separated by �2 kb ofURA3 sequence in opposite orien-tation generates noncomplemen-tary breaks that are preferentiallyjoined by Ku- and Rad52-independent MMEJ. The locationof primers for PCR amplificationand sequencing are shown by ar-rows. (B) Survival frequency afterinduction of HO breaks of eachmutant strain (shaded bars) andthe corresponding KU70 deletionderivatives (solid bars). The fre-quency of survival after an HO-induced DSB was calculated bydividing the number of coloniesgrowing on YEP–GAL by the num-ber of colonies growing on YEPD.Each value represents the averagefrom at least three independentexperiments 6 standard devia-tion. (C) Junctional sequencesobserved in survivors of SLY19and the mutant derivatives wereused to calculate the frequencyof repair events carried out byMMEJ or NHEJ. Typically, MMEJinvolves .5 bp of imperfectmicrohomology, whereas NHEJjoins ends using ,5 nt of comple-mentary base pairing. The regionspanning junctional sequenceswas PCR amplified with a set ofprimers ½p2 and pX (A)� that an-

neal 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).


endonuclease activities (Paull and Gellert 1998;Trujillo and Sung 2001) and a DNA hairpin endonu-clease activity that can incise DNA at a single-strand/duplex junction (Paull and Gellert 1999; Trujillo

and Sung 2001). We thus reasoned that Mre11 func-tions to resect DNA ends in the MMEJ reaction. Shouldthe Mre11 complex provide the nuclease activity inMMEJ, we predicted that the mre11 mutations that inac-tivate the nuclease activity, such as the mre11-H125N(Moreau et al. 1999) and mre11-3 mutations (Bressan

et al. 1998), will also impair MMEJ. The mre11-H125Nmutant has been confirmed biochemically to be defi-cient for nuclease activity but maintains the ability toform a heterotrimeric complex with Rad50 and Xrs2(Moreau et al. 1999).

The mre11 gene deletion derivative of SLY19 (SLY19-mre11D) was transformed with centromeric plasmids

bearing mutant mre11 constructs to assess the effect ofthe mre11 mutations on cell survival after induction ofpersistent HO cleavage by plating cells onto galactose-containing medium (Figure 3A). The results were com-pared to SLY19-mre11D transformed with either thewild-type MRE11 expression construct or an empty vec-tor. We also sequenced �100 PCR-amplified repairjunctions from the survivors to ensure the repair eventswere mediated by 5 bp or more of microhomology,typical of MMEJ products. We found that expression ofthe nuclease defective mre11-H125N or mre11-3 muta-tions rescued lethality of the mre11 null mutant uponDSB induction (Figure 3A). However, analysis of repairjunctions from survivors revealed that almost all wererepaired by Ku-dependent NHEJ rather than MMEJ(Figure 3A and supplemental Table 2 at http://www.genetics.org/supplemental). Expression of an all butnull allele of mre11-P162S (Chamankhah et al. 2000)markedly reduced cell survival from HO breaks both byMMEJ and NHEJ. Our interpretation of these results isthat the nuclease activity of Mre11 is critical for MMEJ.

Overproduction of the 59 to 39 exonuclease Exo1 waspreviously shown to complement the resection defect ofthe 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 themre11D strain (Tsubouchi and Ogawa 2000). If Mre11participates in MMEJ as a nuclease to create single-stranded DNA (ssDNA) at DSBs, Exo1p may rescue theMMEJ deficiency of the mre11D strain. We overexpressedEXO1 in SLY19-mre11D and assayed the MMEJ defect ofthis strain by measuring survival after HO cleavage andcataloging the repair events from sequencing the repairjunctions. As shown in Figure 3B, overproduction of Exo1but not the vector alone partially but consistently rescuedthe decreased survival of SLY19-mre11D. In addition, anmre11D exo1D double mutant showed a further reductionin survival after HO expression rather than the mre11D

mutant alone (Figure 3B). We thus propose that onefunction of the Mre11 complex in MMEJ is degrading59 DNA ends to generate ssDNA to reveal microhomol-ogy for annealing. This suggests that in the absence ofMre11, other nuclease(s) such as Exo1 can provide lessefficient end resection for the limited MMEJ reactions.

Rad1/Rad10 and Srs2 are involved in MMEJ: Rad1and Rad10 form a heterodimeric structure-specific en-donuclease that removes 39 flaps during HR (Sugawara

et al. 1997). Rad1/Rad10 likely participates in MMEJ bycatalyzing 39 flap removal after annealing of microho-mology between ssDNA (Ma et al. 2003). Consistent withtheir function as a complex, a yeast strain deleted forboth RAD1 and RAD10 showed identical MMEJ de-ficiency as the rad1D and rad10D single mutant strains(Figure 2, B and C).

Srs2 suppresses recombination by displacing Rad51from ssDNA (Krejci et al. 2003; Veaute et al. 2003). Srs2

Figure 3.—The role of Mre11 nuclease activity in MMEJ.(A) SLY19-mre11D was transformed with yeast centromeric plas-mids expressing MRE11, mre11-H125N, mre11-3, mre11-P162S,or an empty vector. Survival frequency and the relative contri-bution of MMEJ and NHEJ to survival after induction of HObreaks were determined by analysis of junctional sequencesobserved in survivors. (B) Survival frequency of SLY19-exo1D,SLY19-exo1D mre11D, or SLY19-mre11D expressing additionalExo1 from a GAL promoter. An empty vector containingthe GAL promoter was used as a control. Each value repre-sents the average from at least three independent experi-ments 6 standard deviation.


also performs prorecombination functions during alle-lic recombination and SSA (Sugawara et al. 2000; Ira

et al. 2003). In SSA, Srs2 may stabilize homologous jointsand promote the removal of 39 flap DNA with the Rad1/Rad10 complex by relieving superhelical stress fromunwinding donor duplex DNA (Paques and Haber

1997). Alternatively, Srs2 can promote MMEJ by displac-ing Rad51 and thus inhibiting recombination of resectedDNA ends. To distinguish between these two possibili-ties, we deleted RAD51 in an srs2D strain and investi-gated its ability to undergo MMEJ repair of an HObreak. We found that deletion of RAD51 fully rescuedthe MMEJ repair defect in srs2D (Figure 4). This resultsuggests that Srs2 is involved in MMEJ as an antirecom-binase by displacing Rad51 from ssDNA.

Polymerase activity of Pol4 is essential for gap fill-inafter annealing between microhomology: MMEJ likelydepends on DNA polymerase(s) to fill gaps of varioussizes ranging from a few to several hundred nucleotidesfollowing 59 nucleolytic degradation to expose micro-homologies within the regions flanking a DSB (Figure1). In addition to Pol4, we tested two other nonessentialpolymerases (Rev3 and Rad30), Pol32 (a subunit ofPold), and poly (A) RNA polymerase Trf4 (Holbeck

and Strathern 1997; McDonald et al. 1997; Gerik

et al. 1998; Wang et al. 2000; Zhao et al. 2004, 2006), fortheir role in MMEJ by determining survival of single-deletion mutant strains following HO expression (Fig-ure 5A). We found that deletion of POL4, POL32,RAD30, or REV3 all led to a 3- to 10-fold reduction insurvival from the HO break because of a combined de-ficiency in NHEJ and MMEJ (pol4D) or the MMEJ defectonly (pol32D, rad30D, and rev3D). Yeast strains lacking

TRF4 did not confer any detectable MMEJ deficiency.Potential involvement of multiple polymerases in MMEJprompted us to consider redundancy among polymerasesin the gap-filling step of MMEJ. We tested this idea byconstructing yeast strains with two or more polymerasegene deletions and assessed their MMEJ deficiency byanalyzing repair events following HO cleavage. None ofdouble mutant strains showed additional MMEJ defi-ciency beyond that of each single gene deletion mutant(Figure 5B). However, deletion of POL32, RAD30, andREV3 together almost completely wiped off any residualMMEJ among survivors (Figure 5B). The results thusreveal redundancy among these polymerases in MMEJrepair.

Pol4 is not a processive polymerase (Bebenek et al.2005). It mostly fills small gaps without synthesizing longstretches of nucleotides (Daley et al. 2005a). Lack ofprocessivity led us to consider the possibility that Pol4plays a role in MMEJ separate from its DNA polymeri-zation activity. Previously, the BRCT domain of Pol4 wasshown to interact with the Dnl4/Lif1 complex and tocouple the gap filling and ligation reactions during Ku-dependent NHEJ (Tseng and Tomkinson 2002, 2004).Consistent with the partial dependency of MMEJ on Dnl4,we reasoned that Pol4 may facilitate the ligation step ofMMEJ by recruiting Dnl4/Lif1 to the repair site. To testthis hypothesis, a yeast centromeric plasmid expressingeither the BRCT domain-truncated pol4 (pol4Dbr) mu-tation or the polymerase-dead pol4-D367E mutation(Wilson and Lieber 1999) was introduced into thePOL4-deleted SLY19 derivative. Expression of pol4Dbrfailed to rescue MMEJ repair in pol4D, suggesting thatthe BRCT domain is required for repair by MMEJ (Fig-ure 5C). More importantly, expression of the polymerase-defective pol4-D367E mutation partially rescued thelethality of an HO break in the pol4D mutant by elevatedKu-dependent NHEJ but failed to recover the MMEJdeficiency. Analysis of repair events among survivors ofHO breaks confirmed that most survivors repaired DSBsby NHEJ via 2 bp of microhomology, deleting 5 nt at thejunctions without DNA synthesis (see supplemental Table2 at http://www.genetics.org/supplemental). These re-sults support the hypothesis that Pol4 is required for gapfilling in MMEJ.

Sae2 and Tel1 promote MMEJ but inhibit NHEJ:Sae2, along with the Mre11 complex, is required forcleavage of Spo11 bound to DNA ends to initiate meio-tic recombination (McKee and Kleckner 1997; Prinz

et al. 1997). Sae2 also contributes to SSA between directrepeat sequences by promoting the nuclease activity ofthe Mre11 complex and tethering broken DNA ends inmitotic cells (Clerici et al. 2005). Interestingly, deletionof SAE2 or expression of a nuclease defective mre11 allele(mre11-H125N) results in a very similar phenotype—an increase in Ku-dependent NHEJ but reduced MMEJ(Figures 2, B and C and 3A). We questioned whether Sae2contributes to MMEJ by promoting Mre11-dependent

Figure 4.—Lack of Rad51 rescues the MMEJ deficiency ofsrs2D. Survival frequency following induction of HO breaks ofsrs2D, rad51D, and srs2D rad51d strains (shaded bars) and thecorresponding KU70 deletion derivatives (solid bars) were de-termined as described in Figure 2. The average from at leastthree independent experiments 6 standard deviation isshown.


nucleolytic degradation but inhibiting Ku-dependentNHEJ. If this is the case, we predict that overexpressionof EXO1 may partially rescue the MMEJ deficiency ofsae2D or sae2D mre11D and simultaneously reduceNHEJ. Increased expression of EXO1 had no impacton MMEJ or NHEJ in sae2D but significantly increasedsurvival of sae2D mre11D mutants upon HO inductionwith a modest increase in MMEJ repair (Figure 6B). Theresults thus support the model that Sae2-dependent 59

resection plays a regulatory role in modulating MMEJand NHEJ.

Sae2 is a phosphoprotein whose phosphorylation de-pends on the Mec1/Tel1 kinases (yeast ATR and ATMhomologs, respectively) and DNA damage (Baroni et al.2004). To test the effect of phosphorylation of Sae2 onMMEJ, we deleted TEL1 or MEC1/SML1 from SLY19and measured their effect on MMEJ efficiency followingtwo HO breaks. We discovered that there was a fivefoldincrease in NHEJ compared to wild type, but that rareMMEJ repair of two simultaneous HO breaks was re-duced in the TEL1-deleted strain (Figure 6A). Thesedata suggest that Sae2 and Tel1 constitute an importantcontrol for antagonistic modulation of MMEJ andNHEJ.

We also assessed the role of Sae2 and Tel1 in NHEJ byexamining the effect of a sae2 or tel1 deletion in SLY1and SLY18 strains (Figure 6, C and D). Repair of a sin-gle HO break in these two strains occurs primarily byKu-dependent NHEJ. We found that deletion of SAE2or TEL1 improved cell survival up to eightfold byKu-dependent NHEJ (Figure 6, C and D). The resultssuggest that suppression of NHEJ by Sae2 is not uniqueto SLY19 and is independent of the MMEJ pathway.

Increase in NHEJ efficiency at the expense of de-creased MMEJ from these mutants after persistent HOcleavage may be explained by the reduced 59 resection(Clerici et al. 2005; Mantiero et al. 2007), as the extentof nucleolytic degradation may be an impediment forNHEJ (Ira et al. 2004). We thus determined the rate ofresection at the DSB from sae2D or tel1D strain by mea-suring the level of replication protein A (RPA) binding,a trimeric single-strand binding protein, to ssDNAformed at or near the DSB with ChIP assay followed byqPCR using primer sets annealed to multiple regionsat the MAT locus (Shim et al. 2005). We found thatdepletion of SAE2 or TEL1 reduced the replicationprotein A (RPA) binding to the DSB and thus decreasedthe formation of ssDNA (Figure 6, E–H).

Figure 5.—MMEJ requires multiplepolymerases including two translesionbypass polymerases. Survival frequency(A) and the contribution of MMEJand NHEJ to repair (B) among survivorsafter induction of HO breaks of strainslacking one or more nonessential DNApolymerases or KU70 were determinedby analysis of junctional sequences ob-served in survivors. (C) Plasmids ex-pressing POL4 or its mutant derivativeswere introduced into SLY19-pol4D, andthe frequency of MMEJ and NHEJ fol-lowing induction of HO breaks wasdetermined as in A. Each value repre-sents the average from at least three in-dependent experiments 6 standarddeviation.


DISCUSSION

To assess the mechanistic overlap between MMEJ,NHEJ, and SSA, we surveyed the majority of knownNHEJ or SSA factors for their roles in MMEJ repair oftwo DSBs lacking complementary base overhangs. Theanalysis newly identified genes responsible for MMEJ.Those include NEJ1, POL4, POL32, RAD10, RAD30, REV3,SRS2, SAE2, and TEL1. In addition, this study providedsome insight into their specific functions in MMEJ. We

also found that nucleolytic degradation suppresses NHEJ,whereas it is essential for MMEJ. We propose that MMEJis achieved by a series of distinct but mechanisticallysimilar biochemical reactions that also occur in NHEJand SSA.

Role of ssDNA formation at DNA DSBs in MMEJand NHEJ: MMEJ requires the Mre11 complex to repairtwo nearby DSBs without complementary end sequen-ces (Ma et al. 2003). By examining the effect of mre11mutations, which impair various enzymatic functions of

Figure 6.—Roles of Sae2, Mec1, and Tel1 in MMEJ and NHEJ. Contribution of MMEJ and NHEJ to repair among survivors afterinduction of HO breaks of sae2D, tel1D, and mec1Dsml1D strains in A or sae2D and sae2D mre11D expressing additional Exo1 in B weredeterminedasdescribed inFigure2.Survival frequency following inductionofHObreaks in sae2Dor tel1DderivativesofSLY1 inCandSLY18 in D,which carry oneor twoHO cleavage sites indirect repeatorientation in the MAT locus, weredetermined byplatingcells onYEP–GAL normalized by the plating efficiency on YEPD. Each value represents the average from at least three independent experi-ments 6 standard deviation. HO cleavage sites in the MAT locus in SLY1 or SLY18 are shown schematically above each graph. In E–HChIP 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 theindicated time after galactose addition, crosslinked with formaldehyde, and fragmented by sonication. After immunoprecipitationandreverse 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 Hto the DSB, as well as primers specific for the PRE1 gene situated on chromosome V as a control. PCR signals from each primer set atdifferent durations of HO expression were quantified and plotted as a graph. IP represents the ratio of the RPA PCR signal before andafter HO induction, normalized by the PCR signal of the PRE1 control. The positions of HO cut site and the location of primers areshown in E. Each point is the average of two separate experiments.


the wild-type protein, we discovered that the nucleaseactivity of the Mre11 complex is critical for MMEJ. Theimportance of Mre11’s nuclease activity in MMEJ isfurther supported by the observed partial complemen-tation of MMEJ deficiency in mre11D mutant cells byoverproduction of yet another nuclease Exo1p. Dele-tion of SAE2, a key component of meiotic DSB process-ing and mitotic SSA, or TEL1 also led to reduced MMEJrepair of two simultaneous DSBs. In addition, reducedMMEJ in the mre11D sae2D mutant is partially rescued byexpressing EXO1 in excess. Together, these data providecompelling evidence that end processing by the Mre11complex and Sae2 is a critical part of the MMEJ reaction.We therefore reasoned that 59 resection activity shoulduncover microhomologies at either side of the DSB forannealing during the MMEJ reaction.

Although it is an important feature of the MMEJrepair reaction, 59 resection actually suppresses NHEJ,as the nuclease defective mre11 mutation or a SAE2 dele-tion greatly stimulated Ku-dependent NHEJ. Previously,unprocessed DNA ends were identified as a preferredsubstrate for NHEJ (Frank-Vaillant and Marcand

2002). NHEJ is most active in the G1 phase of the cellcycle when the nucleolytic processing of DNA ends isless active (Ferguson et al. 2000; Aylon et al. 2004; Ira

et al. 2004). Together, these results suggest that nucle-olytic end processing affects both NHEJ and MMEJ butin opposite ways. It promotes MMEJ but limits NHEJreactions. The inhibitory effect of 59 resection on NHEJsuggests that yKu may be inhibitory to the MMEJ pro-cess. The absence of Ku accelerates nucleolytic degra-dation (Liang and Jasin 1996; Lee et al. 1998) and canstimulate the MMEJ reaction. The results also explainwhy two DSBs with no complementary end sequencesare preferentially repaired by MMEJ rather than byNHEJ (Ma et al. 2003). The lack of complementary basesprevents DSB ends from annealing, making them morevulnerable to nuclease activity.

The Mre11 complex and Sae2 are modified by theMec1 or Tel1 kinases upon DNA damage (Usui et al.2001; Baroni et al. 2004). Loss of Tel1 (and to a lesserextent Mec1) increased NHEJ but reduced MMEJ. Inter-estingly, Sae2 phosphorylation was shown to be criticalfor processing and displacing Mre11 from DSB ends(Clerici et al. 2005, 2006). Considering the role of nu-cleolytic degradation in MMEJ and NHEJ, it seems logi-cal to propose that phosphorylation of Sae2 modulatesMMEJ by promoting DSB-induced DNA end resection.

Role of Srs2 and Rad1/Rad10 in MMEJ: Following 59

degradation of DSB ends to generate 39 ssDNA, propermicrohomologies anneal, and the 39 flaps are removedfor gap fill-in synthesis and ligation to complete MMEJ.We showed that both Rad1 and Rad10 are needed forMMEJ but dispensable for NHEJ. As Rad1 and Rad10form an endonuclease complex that removes 39 flapsduring gene conversion or SSA (Fishman-Lobell andHaber 1992; Davies et al. 1995; Ferreira and Cooper

2004), we propose that this complex catalyzes 39 flapremoval in MMEJ.

We demonstrated that deletion of SRS2 displayed re-duced MMEJ frequency, which can be reversed by simul-taneous deletion of RAD51. We imagine that ssDNAformed by Mre11-dependent 59 resection of DSBs willattract binding of single-strand binding proteins such asRPA or Rad51. Given its function as a strand-annealingprotein between ssDNA and duplex DNA molecules(Sung 1994), Rad51 binding to ssDNA may interferewith annealing between single-stranded DSB ends dur-ing SSA or MMEJ. The inhibitory effect of Rad51 in SSAis supported by the finding that deletion of RAD51significantly improves the efficiency of SSA (Ira andHaber 2002). Alternatively, Rad51 may interfere withbinding of yet another single-strand binding proteinthat catalyzes annealing of ssDNA. Genetic and biochem-ical evidence strongly suggests that Srs2 is an antire-combinase, displacing Rad51 from ss- or double-strandedDNA (dsDNA) molecules (Krejci et al. 2003; Veaute

et al. 2003). We propose that Rad51 displacement fromssDNA is necessary for efficient MMEJ, and Srs2 achievesthis through its Rad51 displacement activity.

Multiple polymerases fill in gaps during MMEJ: Ana-lysis of MMEJ repair events suggests that cells frequentlyuse microhomologies, with one only a few bp away butthe other at 60 to 300 bp away from break. Provided that59 resection exposing microhomology proceeds sym-metrically at either side of the break, MMEJ needs con-siderable DNA synthesis to fill these gaps for efficientMMEJ. Interestingly, we found that at least three DNApolymerases and one accessory factor of a major repli-cative polymerase are needed to accomplish efficientMMEJ. Why does MMEJ require multiple polymerases?One reason may be that each polymerase contributes toMMEJ at different cell-cycle phases, locations, or se-quences. Indeed, Rad30 and Rev3 expression fluctuatesduring the cell cycle (Saccharomyces Genome Database,http://www.yeastgenome.org), whereas expression ofPol4 is constant throughout all phases. Alternatively,the requirement for multiple polymerases may reflectthat MMEJ is achieved by the ‘‘two polymerase two stepmodel’’ originally proposed to account for multiplepolymerase involvement in translesion synthesis (Wang

2001). According to this model, one or more poly-merases initiate gap synthesis, followed by extensionsynthesis catalyzed by yet another polymerase(s). Pol4 iswell suited to initiate 39-end synthesis from mismatchesat annealed microhomologies because of its relaxedtemplate dependency (Pardo et al. 2006). However, thelow processivity of Pol4 prevents it from long gap fill-insynthesis. Indeed, recombinant Pol4 demonstrated verypoor processivity in primer extension assays with ,25%of a 5-nt gap filled in (Bebenek et al. 2005). Pold, Rad30,Rev3, or yet another polymerase(s) may contributeto MMEJ by extending synthesis beyond the initialsynthesis by Pol4. As deletion of Pol4 did not completely


eliminate MMEJ, we also propose that another poly-merase may initiate gap synthesis even in the absenceof Pol4.

Among four polymerase mutants tested, pol32D is mostdefective in MMEJ. The results thus suggest that Pol32plays the most critical role(s) among polymerases inMMEJ, and its function cannot be replaced by otherpolymerases. As one of the Pold subunits, the majorcontribution of Pol32 in MMEJ may simply indicate thesignificance of Pold-mediated DNA synthesis in MMEJ( Johansson et al. 2004). However, Pol32 is uniquely in-volved in UV-induced mutagenesis (Huang et al. 2000)and physically interacts with Srs2, Rad9, or alternateclamp loaders, Rad24, Ctf18, and Elg1 (Bellaoui et al.2003; Tong et al. 2004). Pol32 has been proposed to loadPolz to the damaged sites (Gibbs et al. 2005). Additionalstudies are needed to reveal the precise function(s) ofPol32 in MMEJ.

Is MMEJ a variant of NHEJ or SSA? Having identi-fied additional common gene products that are requiredfor MMEJ, NHEJ, and SSA, one may wonder if MMEJ is avariant of NHEJ or SSA or just a salvage pathway thatcells rarely use rather than a separate repair mechanism.Lack of g-irradiation sensitivity in XPF (mammalianhomolog of yeast Rad1) mutants supports the viewthat MMEJ contributes little to survival from damage(Friedberg et al. 1995). The strong dependence of MMEJon SSA or NHEJ components also suggests that thereare many common biochemical steps between theseprocesses, although they are not identical. However, ourresults provide an alternative explanation as to why thelack of MMEJ does not necessarily lead to an inability torepair DSBs. Reduced MMEJ in several mutants is com-pensated by a concomitant increase in NHEJ efficiency.Remarkable plasticity of MMEJ, which relies on multipleredundant factors for execution of biochemical steps,further complicates simple measurement of its contri-bution in damage tolerance. The consequences of MMEJdeficiency may be underestimated by the apparent lackof damage sensitivity. Additional studies are needed toaccurately measure the consequences of MMEJ deficiency,which includes the cataloguing of repair events thatoccur in the absence of MMEJ repair. In fact, deletion ofBrca1 or Chk2 alters the spectrum of repair events ratherthan dramatically changing the overall efficiency of dam-age repair (Zhong et al. 2002; Zhuang et al. 2006).

We thank Steve Brill, James Haber, Kyungjae Myung, Patrick Sung,and Tom Wilson for reagents and helpful comments. We also thankDebra Walther for editorial assistance. This work was supported byfunds from the National Institutes of Health GM071011 and the pilotaward from the San Antonio Cancer Institute.

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Communicating editor: N. M. Hollingsworth


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