Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.
CHROMOSOMAL DNA double-strand breaks (DSBs) are formed after exposure of cells to various physical and chemical DNA-damaging agents such as ionizing radiation, bleomycin, and methylmethane sulfonate (MMS). DSBs are also produced endogenously through the action of intracellular enzymes and chemicals such as the highly reactive free radicals derived from oxygen metabolism. Recent studies have suggested that DSBs occur frequently in DNA containing specific at-risk sequence motifs (ARMs; e.g., in DNA containing trinucleotide repeats or large inverted repeats; Nag and Kurst 1997; Freudenreichet al. 1998; Gordenin and Resnick 1998) and may also form after induction of replication arrest (Seigneuret al. 1998). Most clastogenic agents (e.g., radiation, MMS, bleomycin, etc.) produce multiple types of DNA damage, but cause cell killing primarily through induction of unrepaired DSBs (Resnick and Martin 1976; Frankenberg-Schwager and Frankenberg 1990; Obeet al. 1992). For example, X-irradiation generates DNA-protein crosslinks, single-strand breaks, and damaged bases and sugars, but only DSBs are strongly correlated with loss of cell viability. DSBs are also generated during cell development. Such enzymatically induced DSBs typically retain undamaged complementary overhangs (e.g., at the ends of DSBs associated with meiotic recombination, mating type switching, and intron homing in yeast; Haber 1992; Muelleret al. 1993; Liuet al. 1995; Belfort and Roberts 1997). More complex termini have been observed at the ends of the defined DSBs that initiate V(D)J recombination in immunoglobulin and T-cell receptor genes during differentiation of cellular immunity in humans (Jeggoet al. 1995; Ramsden and Gellert 1995; Chu 1997). Despite differences in structure and cleavage mechanism, all types of DSBs appear to exert similar effects on chromosome and cellular metabolism, including reduction of cell viability and induction of mutation, recombination, and aneuploidy (Obeet al. 1992; Bryant and Johnston 1993; Strathernet al. 1995; Lewiset al. 1998). In addition, DSBs have been shown to arrest growth at DNA damage-responsive cell-cycle checkpoints and induce transformation of human and animal cells (Weinert and Hartwell 1988; Obeet al. 1992; Nelson and Kastan 1994; Siede 1995).
The repair of DSBs is primarily a function of genes associated with two discrete pathways that are conserved in all eukaryotic organisms from yeast to humans. One pathway, involving repair by homologous recombination, was first described for the repair of radiation-induced DSBs over 20 years ago (Resnick 1976; Resnick and Martin 1976). This model for recombination in mitotic cells remains largely as originally presented (Szostaket al. 1983; Thaler and Stahl 1988; Peteset al. 1991), although additional classes of DSB repair events have been identified (Klein 1995; Malkovaet al. 1996). The second major pathway of DSB repair, nonhomologous end-joining (NHEJ), provides for the direct rejoining of broken molecules and may be either precise or error prone (Jeggoet al. 1995; Boulton and Jackson 1996; Milneet al. 1996; Chu 1997; Hendrickson 1997).
Genes specifically associated with recombinational repair of DSBs in mitotic yeast cells include RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, and the RFA (single-stranded DNA binding protein) complex genes (Haynes and Kunz 1981; Game 1993; Firmenichet al. 1995; Hayset al. 1995; Bai and Symington 1996). rad52 mutants are profoundly deficient in recombination in many assays (Resnick 1976; Rattray and Symington 1995; Sugawaraet al. 1995; Zou and Rothstein 1997), while mutations in RAD51 (a structural and functional homologue of the recA gene of Escherichia coli), RAD54, RAD55, RAD57, or RAD59 often produce less severe deficiencies. In addition, Rad51, Rad52, Rad54, Rad55, and Rad57 (involved in strand annealing and exchange) appear to form distinct protein:protein associations in vivo (Bai and Symington 1996; Cleveret al. 1997; Golubet al. 1997; Sung 1997a,b; Bensonet al. 1998; Newet al. 1998; Shinohara and Ogawa 1998).
The efficiency of homologous recombination is influenced by several additional genes associated with DSB repair. For example, RAD50, MRE11, and XRS2 participate in NHEJ (described below) and are not required for mating type switching or interchromosomal recombination; however, each of these genes is required for resistance to ionizing radiation (Saekiet al. 1980; Haynes and Kunz 1981; Game 1993; Hendrickson 1997) and a functional RAD50 gene is necessary for efficient homologous intrachromosomal recombination in some assays (Rattray and Symington 1995; Tranet al. 1995; Elias-Arnanzet al. 1996). In addition, mutations within the genes encoding DNA helicases Hpr5(Srs2), Rad5, and Rdh54 (Lawrence 1994; Milneet al. 1995; Schild 1995; Chanetet al. 1996; Ahneet al. 1997; Klein 1997; Shinoharaet al. 1997), the exonuclease Exo1 (Fiorentiniet al. 1997), excision repair endonuclease Rad1/Rad10 (Habrakenet al. 1994; Rodriguezet al. 1996; Paques and Haber 1997), mismatch repair proteins Msh2 and Msh3 (Paques and Haber 1997), or Cdc1 (Halbrook and Hoekstra 1994) can exert strong effects on spontaneous and/or damage-induced recombination.
NHEJ repair has been detected in yeast by assessing the efficiency of recircularization of linear plasmid DNA after cellular transformation and through study of infrequent, homology-independent DSB repair events (Schiestlet al. 1994; Boulton and Jackson 1996; Milneet al. 1996; Moore and Haber 1996; Tsukamotoet al. 1997b). Genes involved in recombination-independent NHEJ repair include HDF1(YKU70), YKU80(HDF2), RAD50, MRE11, XRS2, DNL4 (the yeast homologue of human DNA ligase IV), SIR2, SIR3, and SIR4 (Boulton and Jackson 1996; Milneet al. 1996; Hendrickson 1997; Scharet al. 1997; Teo and Jackson 1997; Tsukamotoet al. 1997a; Wilsonet al. 1997). The proteins encoded by most genes involved in NHEJ bind to the ends of linear double-stranded DNA. Past studies (including two-hybrid analyses and immunoprecipitation experiments) have identified Ku70:Ku80, Rad50:Mre11:Xrs2, and Sir2:Sir3:Sir4 complexes within cells (Johzuka and Ogawa 1995; Dolganovet al. 1996; Chu 1997; Hendrickson 1997; Sherman and Pillus 1997; Tsukamotoet al. 1997a). More recent reports have suggested that these complexes have different functions in NHEJ; e.g., Ku heterodimers are associated with bridging of the ends of broken DNA molecules while Rad50 and Mre11 appear to be involved in nucleolytic processing of DNA ends (Sharples and Leach 1995; Moore and Haber 1996; Caryet al. 1997; Hendrickson 1997; Panget al. 1997).
Most yeast genes primarily involved in recombination (RAD51, RAD52, RAD54, RAD55, RAD57, RAD59) or NHEJ (HDF1, YKU80, RAD50, MRE11, SIR2, DNL4) have structural homologues in human cells. Protein:protein interactions have also been conserved, e.g., Rad51: Rad52, Rad51:Rad54, Ku70:Ku80, and Rad50:Mre11 associations have been identified in both yeast and human cells (Johzuka and Ogawa 1995; Shenet al. 1996a; Cleveret al. 1997; Golubet al. 1997; Hendrickson 1997). Conservation of the function(s) of members of each pathway is suggested by biochemical and genetic studies and by reports that expression of human Ku70 and Rad54 can complement repair defects in yeast hdf1(yku70) and rad54 mutants, respectively (e.g., Kanaaret al. 1996; Barnes and Rio 1997; Bezzubovaet al. 1997). In addition to their roles in DSB repair, genes involved in recombination and NHEJ have also been implicated in normal cell development and in processes leading to human pathology. For example, human Ku70 and Ku80, in conjunction with an associated DNA-dependent protein kinase (DNA-PK), are required for V(D)J recombination events that occur in immunoglobulin and T-cell receptor genes during development of immunity (Jeggoet al. 1995; Chu 1997). In addition, deletion of the mouse RAD51 recombinase gene produces an embryonic lethal phenotype (Lim and Hasty 1996; Tsuzukiet al. 1996). Furthermore, recent experiments have established that human Rad51 interacts with the tumor suppressor proteins encoded by p53, BRCA1, and BRCA2 in vivo (Shen et al. 1996a,b; Sturzbecheret al. 1996; Buchhopet al. 1997; Mizutaet al. 1997) and that sequence polymorphisms in human KU80 (XRCC5) and XRCC3 (a RAD51 homologue) are associated with tumorigenesis (Priceet al. 1997).
Because traditionally employed clastogens (X rays, MMS, bleomycin, etc.) produce a variety of chromosomal DNA lesions in addition to DSBs, systems for studying the genetic and cytological consequences of expression of site-specific endonucleases (which produce complementary or blunt-ended DSBs only) have been developed in yeast and mammalian cells (e.g., Obeet al. 1992; Bryant and Johnston 1993; Godwinet al. 1994; Bennettet al. 1996; Sargentet al. 1997; Lianget al. 1998). Studies in yeast have analyzed the repair of DSBs induced by HO, EcoRI, and I-SceI (Barnes and Rine 1985; Haber 1992; Plessiset al. 1992; Barnes and Rio 1997; and see references in Lewiset al. 1998).
We have recently employed yeast strains that permit modulation of GAL promoter induction kinetics to show that EcoRI-induced DSBs arrest cell growth at the G2/M transition and stimulate interchromosomal recombination, but do not cause more cell killing in rad52 mutants than in wild-type cells (Lewiset al. 1998). However, expression of EcoRI was lethal in hdf1 and rad9 mutants, suggesting separable roles for end-joining, RAD52-mediated recombination, and DNA damage-responsive checkpoint pathways in the repair of endonuclease-induced DSBs.
In this study we demonstrate that many genes associated with NHEJ are essential for repair of chromosomal DSBs in vivo. Although several recombinational repair genes were required for progression of cells past the G2 checkpoint, only NHEJ genes were required for survival after EcoRI-induced cleavage of chromosomal DNA. In addition, analysis of cellular responses suggested that the DSB repair functions of several end-joining pathway genes, e.g., HDF1/YKU80 vs. RAD50/MRE11/XRS2 or SIR2/SIR3/SIR4, are genetically separable. The complementary-ended DSBs produced by the endonuclease are structurally similar to DSBs generated during normal cell development (i.e., during meiosis, mating type switching, and site-specific homing of intron DNA), which suggests that such DSBs might also be repairable by both recombination and NHEJ pathways.
MATERIALS AND METHODS
Genetic methods and media: Yeast growth media, including YPD, YPG, synthetic dropout, and sporulation plates were prepared as described (Sherman 1991). Transformation of yeast cells was accomplished using a modification of the lithium acetate procedure (Gietzet al. 1995). For selection for G418 resistance, YPD plates containing 0.2 mg/ml G418 (Life Technologies) were prepared as described (Wachet al. 1994). 5-fluoroorotic acid (5-FOA)-resistant colonies were isolated using synthetic plate media containing 0.1% 5-FOA (American Biorganics, Niagara Falls, NY). All strains were propagated and assayed at 30° except Δhdf1 mutants, which were propagated at 25° and assayed for DNA repair at 30°. A 137cesium source was employed at a dose rate of 2.75 krads/min for analyses of cellular growth responses to ionizing radiation.
Strain construction: Yeast strains constructed for this study are listed in Table 1. All strains are derivatives of the A364-based strain 334 (Hovlandet al. 1989). This strain contains the mutant allele reg1-501. GAL1 and GAL10 promoter activity in reg1-501 cells is low in glucose media, but addition of galactose induces the GAL1/10 promoters while cells continue using glucose as carbon source. We note that although GAL transcription is apparently quite low in reg1-501 strains in glucose media (Hovlandet al. 1989; Niederacher and Entian 1991; Lewiset al. 1998), basal levels of expression in Δreg1 host strains remain to be established. Strain 334 (and T334, the Δtrp1 derivative used here) also contains a mutation in the GAL1 gene that blocks metabolism of exogenous galactose. Plasmids used for gene disruption included p52LEU (Δrad52:: LEU2) and pΔrad1Blast (Δrad1::hisG-URA3-hisG) obtained from E. Perkins, pNKY83 (Δrad50::hisG-URA3-hisG) obtained from N. Kleckner, pSL101 (Δrad54::hisG-URA3-hisG) and pSTL11 (Δrad55::LEU2) from L. Symington, pAM50 (Δrad51:: LEU2) and pSM51 (Δrad57::LEU2) from D. Schild, and pΔ21 (Δrad6::hisG-URA3-hisG) and pDM610.23 (Δsir4::LEU2) obtained from C. Bennett.
Deletions of rad2, rad5, rad27, rdh54, exo1, hpr5(srs2), xrs2, and mre11 were accomplished using PCR-mediated disruption as described (Baudinet al. 1993; Wachet al. 1994; Lewiset al. 1998). rad50 rad52 and xrs2 rad52 double mutants were created by deleting RAD50 or XRS2 in YLKL351 (Δrad52::LEU2, Δhis3::GAL1::EcoRI) cells containing the plasmid YcpRAD52 (RAD52, URA3). Gene disruption/deletions were confirmed by PCR analysis of genomic DNA and/or by genetic crosses except for rad1 and rad2 strains, which were identified by their sensitivity to ultraviolet light. Two or three independent isolates of each gene disruption were analyzed in the experiments described below. Nucleotide sequences of primers used for deletion and PCR confirmation are available upon request.
Endonuclease-induced changes in cell growth and viability: Growth of cell cultures was monitored by counting cells using a hemacytometer. Cell survival after induction of endonuclease expression in galactose was calculated as the number of viable cells per milliliter observed on YPD plates divided by the number of cells per milliliter in the culture determined by hemacytometer. YPD broth containing 3% glucose was used for overnight cultures and for control time-course experiments. Earlier studies using the reg1-501 strain employed YPD containing 2% glucose (Hovlandet al. 1989; Lewiset al. 1998), but this was modified after determination that prolonged growth of rad9 strains containing integrated GAL1:: EcoRI in standard YPD media (for 12-24 hr) led to glucose depletion and apparent modest induction of the GAL1 promoter (Lewiset al. 1998). Inducing media consisted of YPD broth with 3% glucose and 2% galactose (YPD + Gal). Haploid cell assays were performed by shifting logarithmically growing cell cultures from YPD to YPD + Gal media at a density of 2-4 × 105 cells per milliliter. For the analysis of wild-type and mutant diploid cells depicted in Table 2, eight independent cultures were used to calculate cell viabilities and standard deviations. Standard deviations were ≤15% of the mean for all other cell survival assays. Diploid strains used for the assays in Table 2 included YLKL350 × YLKL313 (Rad+), YLKL372 × YLKL397 (rad50), YLKL381 × YLKL439 (rad51), YLKL351 × YLKL423 (rad52), and YLKL407 × YLKL433 (mre11). Each of the diploid strains contains a single integrated Δhis3::GAL1:: EcoRI fusion.
Distribution of cells in G1, S, and G2/M: Cell-cycle progression was monitored as described (Lewiset al. 1998). Large-budded cells were defined as cells in which the bud was >50% of the size of the mother cell. For analysis of unbudded, small-budded, and large-budded cells after 12 hr of induction in galactose media, 300-400 cells in each culture were counted.
Survival of cells expressing EcoRI requires genes associated with NHEJ, but not recombinational repair: We previously demonstrated that expression of EcoRI endonuclease inhibits growth and induces G2 arrest in rad52 strains, but does not produce significantly more killing than in wild-type cells (Lewiset al. 1998). This work, like the previous study, has employed the reg1-501 strain T334 (Hovlandet al. 1989; Niederacher and Entian 1991). GAL1-GAL10 promoter activity is repressed in glucose media in this strain, but is induced in media containing glucose plus galactose. The ability to induce GAL promoter activity without changing carbon source is advantageous because cell growth rates and the relative lengths of each cell-cycle phase are different in cells metabolizing glucose vs. galactose (Barford and Hall 1976).
The observation that RAD52 was required for progression of cells past the checkpoint at G2, but not for survival, indicated that repair of EcoRI-induced DSBs involves additional genes. The effects of EcoRI expression on cell viability in strains containing deletions in several additional RAD52 group genes are described in Figure 1, A-C. All cells used in the experiments contain a chromosomal Δhis3::GAL1::EcoRI fusion. Assays were performed using logarithmically growing cells as previously described (Lewiset al. 1998) with slight modification (see materials and methods). The rad52 strain YLKL351 (Lewiset al. 1998) is included in Figure 1 for comparison. Strains containing deletions in genes specifically associated with strand annealing and exchange in mitotic cells (RAD51, RAD52, RAD54, RAD55, and RAD57) displayed high survival throughout the time course (Figure 1A).
rad50, mre11, and xrs2 mutants previously have been shown to be deficient in NHEJ repair using plasmid recircularization assays, but are not defective in homologous interchromosomal recombination or mating type switching (gene conversion) in mitotic cells (Boulton and Jackson 1996; Milneet al. 1996; Moore and Haber 1996; Hendrickson 1997). As shown in Figure 1B, continuous EcoRI-induced scission of DNA in these mutants caused extensive cell killing. Plating efficiency was reduced to 2-3% after 12 hr of endonuclease expression.
The SIR2, SIR3, and SIR4 genes, which are required for transcriptional silencing at MAT and in the telomeric regions of yeast chromosomes, are also involved in plasmid DSB repair (Tsukamotoet al. 1997a). The ability of cells containing a deletion of SIR4 to repair chromosomal DSBs induced by EcoRI is depicted in Figure 1C. The mean surviving fraction was reduced to 8% in sir4 mutants, indicating that repair of the cohesive-ended DSBs is impaired in these strains. Also presented in the figure is a comparative analysis of cell survival and cell cycling in the Δhdf1 strain YLKL389. As noted earlier, we previously observed (using slightly different media; see materials and methods) that expression of EcoRI is lethal in hdf1 cells in the T334 strain background. The kinetics of cell killing in Ku-deficient cells was similar to that of rad50, mre11, and xrs2 mutants and plating efficiency was reduced 30-fold after 12 hr (Figure 1C).
The effects of EcoRI-induced breakage of chromosomal DNA on cell-cycle progression are shown in Figure 2, A-C. Recombination-defective mutants arrested growth as large-budded cells (Figure 2A), indicating that they were unable to progress past the G2/M boundary when EcoRI was continuously expressed. Most arrested cells were much larger in size than normal G2 cells. We previously demonstrated that this DSB-induced arrest corresponds to a RAD9-dependent, DNA damage-responsive checkpoint that is phenotypically similar to the damage-induced arrest described by Weinert and Hartwell (1988). Wild-type cultures displayed a modest increase in large-budded cells during the time course. This prolongation of the G2 phase is transient and wild-type cells advance to early stationary phase at 24 and 48 hr (Lewiset al. 1998; data not shown). All of the strains studied in Figures 1 and 2 exhibited high survival and normal cell cycling when grown in YPD (noninducing) media (Figure 1C and data not shown).
Interestingly, the growth of rad50, mre11, and xrs2 mutants was blocked, with most cells arrested at the G2 checkpoint (Figure 2B; ∼70% large-budded cells after 12 hr), but this pause was not sufficient for cells to repair the induced strand breaks after synthesis of the endonuclease was repressed. Most sir4 cells eventually arrested growth in G2 phase, but Δhdf1 cells did not (Figure 2C). Growth of Ku-defective cells was blocked, but these cultures consisted primarily of a mixture of enlarged unbudded and large-budded cells, suggesting that arrest occurred in both G1 and G2 (discussed below).
Requirement for RAD3 and RAD6 group genes after EcoRI-induced scission of chromosomal DNA: The potential involvement of RAD1 and RAD2 (nucleotide excision repair genes in the RAD3 epistasis group; Haynes and Kunz 1981; Habrakenet al. 1994) and RAD5 and RAD6 (RAD6 epistasis group genes involved in postreplication repair and mutagenesis; Haynes and Kunz 1981; Lawrence 1994) in the repair of EcoRI-induced DSBs has also been examined (Figure 3, A-D). rad5 and rad6 mutants are moderately sensitive to ionizing radiation and ultraviolet light, but their functions in the repair of single- or double-strand breaks remains obscure. Survival curves for Δrad5 and Δrad6 strains were comparable to those of wild-type cells (Figure 3A). However, growth of rad5 cells was strongly inhibited and these mutants displayed a strong cell-cycle arrest response that was similar to that of recombination-deficient RAD52 group mutants (Figure 3B). This result suggests a role for RAD5 in the recombinational repair pathway and is consistent with a recent report indicating that recombinational repair of gapped plasmid DNA is greatly reduced in cells lacking the Rad5 DNA helicase (Ahneet al. 1997). The response of rad6 cells was complex. These mutants grew slowly when EcoRI was expressed and accumulated enlarged G2 phase cells only at late time points (12 and 24 hr; Figure 3B and data not shown). These results suggest that repair of the induced DSBs was relatively efficient at earlier time points (4 and 8 hr) when fewer breaks are detectable by gel analysis (Lewiset al. 1998 and data not shown), but that a subset of DSBs required processing by Rad6 at later time points.
The excision repair endonuclease encoded by RAD1/RAD10 plays a role in cleavage of recombination intermediates and in the processing of nonhomologous DSB ends (Habrakenet al. 1994; Rodriguezet al. 1996; Paques and Haber 1997). Furthermore, 5′ single-stranded overhangs similar to those produced by EcoRI have previously been shown to be substrates for cleavage by the Rad2 endonuclease in vitro (Habrakenet al. 1995). The possibility that Rad1/Rad10 or Rad2 might be involved in processing of the ends of EcoRI-induced DSBs has been assessed in Figure 3, C and D. Survival of Δrad2 mutants was not affected by EcoRI, but Δrad1 strains displayed a modest, linear decrease in viability during the time course (Figure 3C). Cell-cycle progression in rad2 mutants was essentially identical to that of wild-type cells. In contrast, rad1 mutants were moderately growth-inhibited and slowly accumulated a high proportion of G2 cells (≥70%) at 12 and 24 hr after induction (Figure 3D and data not shown). These results suggest that a subset of DSBs produced by EcoRI are processed by the Rad1/Rad10 endonuclease, but not by Rad2.
To test the possibility that RAD1/RAD10 or RAD2 nuclease processing of cohesive-ended DSBs becomes more significant in the absence of other repair pathways, i.e., recombination or end-joining, cell survival was also monitored in double mutants. The viability of Δrad1 Δrad52 and Δrad2 Δrad52 cells was reduced ∼7-fold during the time course (Figure 3C). This effect was slightly greater than that seen in Δrad1 and Δrad2 single mutants, but cell killing did not approach levels observed in hdf1, rad50, mre11, or xrs2 mutants (∼20-40-fold killing after 12 hr). The kinetics and magnitude of growth arrest as large-budded cells in each double mutant cell culture appeared similar to that of rad52 cells (Figure 3D).
The data presented in Figures 1, 2, 3 and in past studies (see Lewiset al. 1998 and references within) support the hypothesis that DSB termini containing single-stranded complementary overhangs can be repaired by multiple pathways in vivo. In contrast, repair of damaged ends produced by ionizing radiation is largely dependent on recombinational repair. The negative effects of EcoRI- and radiation-induced DSBs in wild-type and repair-deficient cells have been compared directly in Figure 4. Growth of cells containing a deletion of RAD52 (recombination deficient), RAD50 or MRE11 (partially defective in recombination and NHEJ), and SIR4 (NHEJ deficient) is inhibited by EcoRI. However, gamma-irradiation (30 krads) blocked growth of the Rec- strains (rad52, rad50, mre11), but not Δsir4 cells (Figure 4). Growth of excision-repair-defective rad1 and rad2 strains on plates was largely unaffected by either clastogen.
Viability and checkpoint responses in end-joining and recombination-defective double mutant strains: The experiments described above indicated that cells experiencing EcoRI-induced DSBs require several recombinational repair genes for progression past the G2 checkpoint, but not for survival. In contrast, Ku-deficient strains and RAD52 group mutants involved in end-joining repair (rad50, xrs2, and mre11) displayed extensive cell killing, but had dissimilar arrest phenotypes (compare Figure 2B vs. Figure 2C). The effects of EcoRI-induced cleavage of DNA in strains deficient in both end-joining and recombinational repair are presented in Figure 5. Surprisingly, growth arrest and cell killing kinetics in rad50 rad52 and xrs2 rad52 double mutants were similar to those of rad50 and xrs2 single mutants, respectively (∼2-3% viable cells after 12 hr; Figure 5A and Figures 1B and 2B). Thus, combination of a deficiency in NHEJ with elimination of almost all homologous recombination did not produce additional killing in this assay.
Interestingly, rad51 hdf1 and rad52 hdf1 double mutants could be constructed in the T334-derived strains used here, but the cells grew poorly and displayed several stress phenotypes in both glucose and galactose media. Characteristics included slow growth, reduced plating efficiency, elevated levels of petite mutants, and an increased fraction of dark (presumably lysed) cells upon microscopic examination (data not shown). These effects were not observed in any other single or double mutant created for these studies and their source remains unclear. We note, however, that expression of EcoRI in wild-type, rad52, or hdf1 strains does not produce elevated levels of petite mutants (Barnes and Rine 1985 and data not shown), suggesting that the phenomena are not due to altered GAL1::EcoRI expression. Furthermore, rad52 hdf1 double mutants have previously been reported to have significantly lower plating efficiency than either single mutant (Siedeet al. 1996). Finally, past reports have demonstrated that DNA damage-inducible genes are constitutively activated in rad52 and hdf1 null mutants, presumably because of elevated levels of unrepaired DNA damage (Magaet al. 1986; Barnes and Rio 1997). This effect may be exacerbated in hdf1 rad52 double mutants, which are defective in both major pathways of DSB repair. The results presented in Figure 5A also demonstrated extensive EcoRI-induced cell killing in rad50 hdf1 mutants that was similar in extent to that observed in rad50 and hdf1 single mutants (∼30-fold killing). rad50 hdf1 cell cultures consisted primarily of unbudded and large-budded cells, suggesting that checkpoint activation occurred in both G1 and G2, as observed earlier in hdf1 cultures (Figure 1C and see below).
Past studies have suggested that some or all DNA end-binding proteins that function in NHEJ repair might affect degradation of DSB termini (e.g., Getts and Stamato 1994; Milneet al. 1996; Moore and Haber 1996). The absence of one or more such proteins might lead to cell killing because the DNA ends become susceptible to cleavage by specific cellular endo- or exonucleases. As an initial test of this hypothesis we constructed double mutant strains that are deficient in Rad50-mediated end-joining and that contain a deletion of an endonuclease or exonuclease gene previously implicated in DSB repair. Changes in cell viability and cell cycling in rad1 rad50, rad2 rad50, and exo1 rad50 mutants upon induction of EcoRI are presented in Figure 5, C and D. EXO1 was included because this exonuclease has been shown to be involved in the processing of DNA ends and in intrachromosomal recombination (Fiorentiniet al. 1997). Cell survival was reduced 20- to 40-fold in each double mutant, which is comparable to that seen in rad50 strains (Figure 5C). All double mutants displayed a strong growth arrest response that was analogous to that observed previously in rad50 single mutants. These data suggest that aberrant processing of the ends of DSBs by Rad1/Rad10, Rad2, or Exo1 is not responsible for the killing observed in end-joining defective rad50 mutants.
Distinct cell cycle checkpoint responses in recombination- and NHEJ-deficient mutants: A detailed examination of the distribution of wild-type and mutant cells within the cell cycle in response to EcoRI-induced scission of chromosomal DNA is presented in Figure 6. The proportion of unbudded (G1 phase), small-budded (an approximation of cells undergoing S phase), and large-budded cells (G2/M phase) was quantitated after 12 hr of EcoRI expression. Cultures of all RAD52 group mutants tested, including recombination-defective rad52 strains and end-joining-deficient rad50 cells, consisted primarily of S and G2 phase cells with most cells enlarged and arrested in G2 (Figure 6 and data not shown). rad50 rad52 double mutants (severely deficient in both recombination and NHEJ) also arrested predominantly at G2 with almost all cells in S or G2. Cultures of hdf1 and hdf1 rad50 mutants consisted of approximately equal numbers of G1 and G2 cells (Figure 6). Thus, the unusual biphasic DNA damage response of Ku-defective strains is dominant to that of rad50 mutants (and by extension to mre11 and xrs2 mutants).
Effects of EcoRI expression on cell viability and checkpoint responses in wild-type and repair-deficient diploid cells: Although both haploid and homozygous diploid RAD52 group mutants are hypersensitive to ionizing radiation-induced DSBs, differences in resistance due to ploidy have been observed. For example, diploid rad50 strains are slightly more radio resistant than their haploid counterparts (presumably due to increased recombinational repair of some induced DSBs), but rad51 and rad52 diploids are not (Saekiet al. 1980; Game 1993). The possibility that EcoRI-induced growth inhibition and/or cell killing observed in repair-deficient haploid strains might be altered in diploids (which can undergo recombinational repair throughout the cell cycle) was tested in wild-type cells and in homozygous rad50, rad51, rad52, and mre11 mutants. Analysis of the growth characteristics of diploid cells containing a single GAL1::EcoRI chromosomal fusion revealed significant differences from haploid cells (Figure 7). Wild-type diploid cells containing either Δhis3::GAL1::EcoRI or Δlys2::GAL1::EcoRI fusions (Lewiset al. 1998 and data not shown) did not exhibit a transient G2 arrest response as previously observed in haploid cells. This result suggests that EcoRI-induced DSBs in the diploids were repaired efficiently and did not activate the checkpoint response system. All homozygous mutant diploids arrested growth in G2 phase after induction of EcoRI expression (70-80% of cells after 8-12 hr) with most of the G2 cells greatly enlarged.
Interestingly, uninduced, logarithmically growing rad50, rad51, rad52, and mre11 diploid cells in glucose media contained ∼50% large-budded cells. This is apparent at the zero time point in Figure 7 and in control time-course experiments in which cells were grown in standard glucose media (data not shown). Haploid RAD52 group mutants displayed only a slight increase in G2 cells in glucose media. A previous study has reported that logarithmically growing rad51 diploid cell cultures, but not rad2, rad6, or rad9 diploids, contain elevated levels (52%) of G2 phase cells (Fingerhutet al. 1984). The extended G2 phase suggests that spontaneously occurring DNA lesions are elevated in these cells and/or that the additional chromosomes in diploids increase the likelihood of a lesion triggering the checkpoint monitoring system.
The effects of EcoRI-induced DNA cleavage on cell survival in wild-type and mutant diploid strains are presented in Table 2. Survival of recombination-deficient rad51 and rad52 mutants was similar in both haploid and diploid cells. However, the approximately twofold decrease in viability observed in wild-type haploid strains was abolished in the diploids. The greatest change was observed in rad50 and mre11 cells. Although these strains displayed a strong G2 arrest response, the cell killing in haploid mutants was largely eliminated in diploids. This suggests that, in contrast to ionizing radiation-induced DSBs, the cohesive-ended DSBs could be efficiently repaired by recombination between homologous chromosomes throughout the cell cycle.
The experiments described here, in combination with our prior study (Lewiset al. 1998), have established that many genes previously implicated in recombination-independent end-joining in yeast cells have an essential role in precise repair of DSBs in vivo. The approach presented here has permitted assessment of the repair of DSBs produced in nuclear, chromatin-associated DNA and the effects of such breaks on progression through the cell cycle. Although the NHEJ pathway was not identified in past analyses of cellular sensitivity to X rays and chemical clastogens, the current study has established that recombination and end-joining repair are both efficient processes in yeast and that this efficiency is critically dependent upon the nature of the DSB ends. Specifically, these results indicate that complementary-ended DSBs that are similar in structure to those produced by endonucleases during cellular development are rejoined by both major pathways of DSB repair. Furthermore, the combined analysis of DSB-induced effects on growth, cell cycling, and survival has led to the demonstration of separable roles for genes involved in the NHEJ pathway of repair. A summary of the results described here and in the previous study (Lewiset al. 1998) is presented in Table 3.
Homologous recombination pathway genes are required for efficient progression past the G2/M checkpoint, but not for survival: We have previously demonstrated that expression of EcoRI in yeast cells results in extensive breakage of cellular DNA and stimulates homologous recombination (Lewiset al. 1998). These experiments included the surprising observation that continuous synthesis of EcoRI arrested the growth of rad52 mutants, but did not produce significantly greater killing than in wild-type cells. This result is in direct contrast to previous studies demonstrating that RAD52 is essential for repair of DSBs induced by ionizing radiation and various DNA-damaging chemicals (Resnick and Martin 1976; Saekiet al. 1980; Moore 1982; Game 1993). In this study we have also observed high survival in cells containing a deletion of the gene encoding the Rad51 recombinase (a structural and functional homologue of E. coli RecA) and in cells deficient in RAD54, RAD55, and RAD57 (summarized in Table 3). The similar responses of these mutants are consistent with recent models suggesting that Rad54 and the Rad55/Rad57 heterodimer work in conjunction with the strand annealing and strand exchange activities of Rad51:Rad52 to effect recombination (Cleveret al. 1997; Golubet al. 1997; Sung 1997a,b; Bensonet al. 1998; Newet al. 1998; Shinohara and Ogawa 1998). Each of these mutants exhibits deficiency in most assays of spontaneous and induced recombination (recombination between some direct repeat sequences is an exception), though rad52 mutants are often more deficient than other single or double mutant strains (Rattray and Symington 1995; Sugawaraet al. 1995; Zou and Rothstein 1997).
The EXO1 gene encodes a 5′-3′ exonuclease that has been implicated in both recombination and mismatch repair. A specific role for EXO1 in homologous recombination, but not in NHEJ repair pathways, is supported by a recent study of recombination between direct repeats (Fiorentiniet al. 1997) and by two additional outcomes described here: the phenotypic responses of exo1 mutants to EcoRI cleavage of chromosomal DNA are similar to those of rad52 mutants and the rapid cell killing observed in rad50 mutants is not altered in rad50 exo1 double mutants (Figure 5). In addition to EXO1, we have recently observed that strains containing a deletion of the putative DNA helicases encoded by SRS2 (HPR5) and RDH54 also exhibit strong DNA-damage responses when EcoRI is expressed, but do not display extensive cell killing (Table 3; K. Lewis and M. Resnick, unpublished results). This suggests that RDH54 (a homologue of RAD54 and a suggested member of the SWI2/SNF2 family of DNA helicases; Klein 1997; Shinoharaet al. 1997) and SRS2 (previously implicated in recombinational repair events mediated by RAD51, RAD52, and RAD54 and involved in the processing of DSB termini; Milneet al. 1995; Schild 1995; Chanetet al. 1996; Paques and Haber 1997) participate in the recombinational pathway of repair of EcoRI-induced DSBs.
Although the two principal pathways of DSB repair, NHEJ and homologous recombination, appear to be common to all eukaryotic organisms, the relative importance of each pathway varies across phylogeny. Past studies have suggested that recombinational mechanisms are dominant in S. cerevisiae and that homology-independent pathways account for most DSB repair in higher eukaryotes (e.g., Roth and Wilson 1986; Godwinet al. 1994; Chu 1997). However, a number of recent experiments have revealed that human and animal cells possess a substantial potential for recombinational repair of DSBs (e.g., Carrollet al. 1994; Bezzubovaet al. 1997; Sargentet al. 1997; Lianget al. 1998; Sonodaet al. 1998). Furthermore, recombinational repair pathways in higher eukaryotes are dependent on many of the same genes as yeast. For example, RAD51, RAD52, and RAD54 homologues have been identified in human and animal cells and shown to affect repair of DSBs induced by DNA-damaging agents (Bezzubovaet al. 1997; Esserset al. 1997; Sonodaet al. 1998).
Essential, but separable, roles for genes associated with NHEJ: In yeast cells the prominant role of RAD52-mediated recombination in repair of DSBs induced by radiation and by chemical DNA-damaging agents is well established (Haynes and Kunz 1981; Game 1993). A number of past multigenic analyses of NHEJ in yeast have analyzed predominantly nonconservative rejoining events in vivo (i.e., restriction enzyme-mediated integration of DNA fragments, deletion formation in dicentric plasmids, and formation of insertions and deletions at MAT after HO endonuclease-induced cleavage; Schiestlet al. 1994; Moore and Haber 1996; Tsukamotoet al. 1997b). Other recent studies have suggested that conservative (precise) repair of chromosomal DSBs produced by HO and EcoRI is defective in cells lacking Ku70 or Ku80 (Barnes and Rio 1997; Leeet al. 1998; Lewiset al. 1998). Finally, linear, cohesive-ended plasmid DNA is accurately and efficiently recircularized after transformation into wild-type and rad52 cells, but not in hdf1, yku80, rad50, dnl4, sir2, sir3, or sir4 mutants (Boulton and Jackson 1996; Milneet al. 1996; Scharet al. 1997; Teo and Jackson 1997; Tsukamotoet al. 1997a; Wilsonet al. 1997).
The multigenic analysis performed here revealed that all mutants previously implicated in NHEJ repair pathways (using the above assays) were hypersensitive to killing by EcoRI, but responses to the enzyme were not identical (see Table 3). Cell viability was reduced 20- to 40-fold in rad50, mre11, xrs2, and hdf1 mutants and was reduced ∼10-fold in sir4 strains. Interestingly, growth of all end-joining mutants was blocked by EcoRI expression, but cell cycle arrest responses varied. rad50, mre11, and xrs2 mutants displayed a rapid, rad52-like increase in enlarged G2 cells after induction of EcoRI synthesis. sir4 mutants also accumulated G2 cells, but did so more slowly. In contrast, hdf1 mutants accumulated as distended G1 and G2 phase cells. This unusual phenotype has been described recently for Ku70-deficient cells after exposure to high temperature or endonuclease-induced DSBs (Barnes and Rio 1997). Temperature-sensitive mutants deficient in CDC28 (encoding a cyclin-dependent protein kinase), TOP2 (topoisomerase II), and CKA2 (coding for part of the catalytic subunit of casein kinase II) have also exhibited arrest at both G1/S and G2/M at their restrictive temperatures (Holmet al. 1985; Hannaet al. 1995; Zarsovet al. 1997). Potential correlations between the mechanisms responsible for biphasic arrest in Ku strains and in the other mutants await further studies. We also note that the similar kinetics of growth and cell-cycle arrest in recombination mutants (which remain viable) and NHEJ mutants (displaying progressive loss of viability) suggests that galactose induction of the endonuclease is comparable in the different mutant strains. This conclusion is also supported by the past demonstration of similar induction kinetics for GAL::HO-induced cleavage at MAT in recombination and end-joining mutants (e.g., see references in Haber 1992; Sugawaraet al. 1995).
The functions of the Ku70:Ku80 heterodimer and Rad50:Mre11:Xrs2 proteins in NHEJ appear to be distinct. The Ku complex has been implicated in the joining reaction for broken DNA ends, but Rad50 and Mre11, which have significant sequence similarity to exoendonucleases encoded by the Escherichia coli SbcC and SbcD proteins, appear to be involved in nucleolytic processing of the ends (Sharples and Leach 1995; Moore and Haber 1996; Caryet al. 1997; Hendrickson 1997; Panget al. 1997). Several phenotypes observed in Ku mutants are also found in rad50, mre11, and xrs2 mutants, but differences that may reflect their disparate roles in NHEJ have also been described. For example, hdf1 and rad50 mutants show equivalent deficiencies in plasmid end-joining, participate in the same pathway of MMS repair based on analysis of epistatic interactions, display unusual processing of dicentric plasmids, exhibit telomere-shortening, and are hypersensitive to killing by EcoRI endonuclease (this work; Boulton and Jackson 1996; Milneet al. 1996; Porteret al. 1996; Siedeet al. 1996; Kironmai and Muniyappa 1997; Tsukamotoet al. 1997b). In addition, Ku and Rad50 proteins have been found associated with the ends of broken DNA in yeast and in human cells (Feldmann and Winnacker 1993; Getts and Stamato 1994; Milneet al. 1996; Nelmset al. 1998). However, a number of phenotypes are unique to Ku-deficient cells. Thus, hdf1 mutants have been reported to be temperature-sensitive for growth, have increased chromosome ploidy, exhibit constitutively elevated transcription of the damage-inducible RNR2 and RNR3 genes, are found associated with replication origin-binding complexes, and have distinct damage-induced growth arrest phenotypes in yeast (this work; Feldmann and Winnacker 1993; Shakibaiet al. 1996; Barnes and Rio 1997). Furthermore, results obtained in this study have indicated that the repair deficiency of Ku mutants is dominant to that of rad50 mutants, as rad50 hdf1 double mutants responded similarly to hdf1 strains (Figure 5). Finally, it is worth noting that several phenotypes of Ku mutants are remarkably strain specific. For example, temperature-sensitive growth at 37°, MMS sensitivity, damage-induced arrest in G1 and G2, and increased radiation sensitivity in rad52 hdf1 double mutants have been described for some strains, but not for others (cited above). Although reports that Hdf1 associates with Sir4 and with replication origin complex proteins in vivo suggest that the phenotypic variability of hdf1 mutants may be due to epigenetic effects, this possibility requires further study (Shakibaiet al. 1996; Sherman and Pillus 1997; Tsukamotoet al. 1997a).
Involvement of RAD3 and RAD6 epistasis group genes in repair of EcoRI-induced DNA damage: Expression of EcoRI in rad1 and rad1 rad52 strains caused an accumulation of cells in G2 and a modest reduction in viability (Figure 3), but cell killing did not approach the levels observed in hdf1 or rad50 mutants. The Rad1/Rad10 excision repair endonuclease cleaves 3′ single-strand overhangs and synthetic Holliday junctions (Habrakenet al. 1994; Rodriguezet al. 1996) and affects processing of nonhomologous DSB ends after cleavage of DNA by HO (Paques and Haber 1997). Unpaired 3′ extensions might be produced in vivo by exonuclease processing of the 5′ overhangs generated by EcoRI. Although 5′ single-stranded overhangs have previously been shown to be substrates for cleavage by the Rad2 endonuclease in vitro (Habrakenet al. 1995), cell cycling and cell survival were not significantly affected by EcoRI expression in rad2 mutants. These results suggest a role for the Rad1/Rad10 endonuclease, but not Rad2, in the processing of EcoRI-induced DSBs in vivo. We note, however, that the cell killing observed in rad50 mutants was not influenced by subsequent deletion of RAD1 or RAD2. Specifically, we found that growth arrest and lethality in rad50 rad1, rad50 rad2, and rad50 exo1 double mutants were not different from that of rad50 single mutants (Figure 5). This indicates that the extensive killing observed when Rad50 is absent is not due to aberrant processing of DSB ends by these three nucleases.
Interestingly, rad5 mutants displayed a rapid, rad52-like G2 arrest phenotype and near-wild-type survival when EcoRI was expressed. A recent report has demonstrated that recombination-mediated gap repair in plasmids transformed into yeast cells is greatly reduced in rad5 mutants (Ahneet al. 1997). This result and the data presented in Figure 3 support a role for the Rad5 DNA helicase in the RAD52 pathway of DSB repair. Expression of EcoRI in rad6 mutants produced a complex phenotype. These strains grew slowly with an approximately wild-type distribution of cells in the cell cycle during the first 8 hr, but with elevated levels of enlarged G2 phase cells 12 and 24 hr after induction. We previously demonstrated that DNA purified from induced Rad+ and rad52 cells contains progressively more unrepaired DSBs after 4, 8, 12, and 24 hr of expression of EcoRI (Lewiset al. 1998). The delayed cell-cycle arrest observed in rad6 mutants suggests that at 4 and 8 hr, when low levels of breaks are present, RAD6-independent pathways are able to repair the damage efficiently. However, at later time points a fraction of the induced DSBs are processed by Rad6.
Endonuclease-induced DSBs produce different effects on cell cycling and viability in haploid and diploid cells: Analysis of the effects of endonuclease expression in diploid cells revealed striking differences between wild-type, recombination-deficient, and NHEJ-defective strains (Table 2). The transient G2 arrest response and small reduction in viability observed during the time course of EcoRI induction in haploid cells were abolished in diploid wild-type cells, but not in rad51 and rad52 diploids. The fact that the loss in viability consistently observed in haploid Rad+ cells was eliminated in diploids suggests that the modest killing effect is due to unrepaired DSBs in haploid G1 cells. Such cells lack sister chromatids for recombinational repair and previously have been shown to be hypersensitive to DSBs (Brunborget al. 1980; Game 1993).
The rapid EcoRI-induced lethality observed in NHEJ-deficient rad50 and mre11 mutants was largely eliminated in rad50/rad50 and mre11/mre11 diploid cells (Table 2). Small and even substantial increases in the ionizing radiation resistance of diploid cells relative to haploids have been observed previously in rad50 mutants, but not in rad51 or rad52 strains (McKee and Lawrence 1980; Saekiet al. 1980; Rao and Reddy 1982; Ivanovet al. 1992; J. L. Nitiss and M. A. Resnick, unpublished results). This increased resistance is generally interpreted to be primarily due to increased recombination capability and we suggest that this is true for repair of EcoRI-induced DSBs in diploids vs. haploids. However, the presence of mating type heterozygosity in the diploids may also contribute to the increased radioresistance of the mutants (Heude and Fabre 1993).
A recent study of plasmid recircularization by Astrom et al. (1999) has suggested that end-joining repair is reduced in a/α diploids relative to a(Δp)/α diploids (containing a deletion of the MATa promoter). It was also demonstrated that the plasmid end-joining deficiency of sir2, sir3, and sir4 haploids could be rescued by blocking expression of the a and α genes from HML, MAT, and HMR. This result implies that SIR genes function indirectly in NHEJ by derepressing expression of the a1/α2 repressor complex. In this study EcoRI-induced DSBs were repaired more efficiently in wild-type a/α diploids than in haploid cells, suggesting that potential defects in NHEJ due to mating type heterozygosity were compensated by the increased capacity for recombinational repair. In addition, cell survival was similar in rad51 and rad52 haploids and diploids, though the latter strains would be predicted to be deficient in both recombination and NHEJ if mating type heterozygosity inhibits end-joining. The simplest interpretation of these data is that the a1/α2 repressor regulates one or more genes involved in rejoining of plasmid DNA ends, but does not play a critical role in end-joining of DSBs in chromatin-associated chromosomal DNA. We are currently investigating this idea.
A schematic representation of the results obtained in this study and in our earlier report (Lewiset al. 1998), based on previously identified DNA repair pathways and protein:protein interactions, is presented in Figure 8. Cell survival after induction of DSBs by EcoRI required gene products implicated in end-joining (Ku70 and Sir4), both end-joining and recombination (Rad50, Mre11, and Xrs2), and DNA damage-responsive checkpoints (Rad9 and Rad17). Our data are consistent with a role for several additional genes (EXO1, HPR5/SRS2, RDH54, and RAD5) in the recombinational pathway of DSB repair. Also shown is DNL4, the yeast homologue of human DNA ligase IV. Cells containing mutations within this gene are not sensitive to commonly used DNA-damaging agents but do display a reduced efficiency of recircularization of linearized plasmid DNA after transformation into yeast cells (Scharet al. 1997; Teo and Jackson 1997; Wilsonet al. 1997). Recent experiments have revealed that expression of EcoRI in T334 strains containing a dnl4::G418 insertion induces a strong G2 arrest response (data not shown), which supports the proposition that this ligase is involved in repair of at least some classes of chromosomal DSBs in nuclear DNA.
Many previous studies have suggested that the efficiency of repair of DSBs is affected by structural differences at the ends of the broken DNA. For example, DSB termini may be damaged (i.e., containing missing or altered bases and sugars as observed at the ends of DSBs produced by ionizing radiation), covalently modified (such as DSBs induced by bleomycin, which can retain phosphoglycolate ester moieties after cleavage), or the ends may be blunt or contain complementary overhangs. DSB ends induced by ionizing radiation have a requirement for RAD52-mediated recombinational repair (Resnick and Martin 1976; Moore 1982; Game 1993).
The experiments described here and in a previous study (Lewiset al. 1998) have now demonstrated that EcoRI-induced chromosomal DSBs, which retain complementary four-base overhangs, can be processed by both the homologous recombination and NHEJ pathways inside cells. This finding is significant because many DSBs that are produced during the course of normal development in eukaryotic cells contain similar end structures. Both HO-induced mating type switching and intron homing (the process by which an intron-containing DNA sequence is inserted into an intronless allele in nuclear and mitochondrial genes) are initiated by formation of a DSB containing four-base complementary ends (Haber 1992; Muelleret al. 1993; Belfort and Roberts 1997). Interestingly, past studies have revealed that recombination occurring in meiosis is initiated by DSBs containing two-base overhangs (Liu and Lichten 1995). Repair of meiotic DSBs has the added complexity that the endonuclease involved in DSB formation, Spo11, forms a transient covalent attachment with one end of the DNA (Keeneyet al. 1997). The results described here suggest that such complementary-ended DSBs may be accurately repaired by distinct groups of proteins active in either homologous recombination or the restitutional mechanism of precise end-joining.
We thank Craig Bennett, Nancy Kleckner, David Schild, and Lorraine Symington for plasmids used in the study. We also thank Vladimir Larionov and Robbert Slebos for comments on the manuscript.
Communicating editor: L. S. Symington
- Received December 23, 1998.
- Accepted April 20, 1999.
- Copyright © 1999 by the Genetics Society of America