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The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells

2001; Springer Nature; Volume: 20; Issue: 22 Linguagem: Inglês

10.1093/emboj/20.22.6540

1460-2075

Laura J. Niedernhofer, Jeroen Essers, Geert Weeda, Berna Beverloo, Jan de Wit, Manja Muijtjens, Hanny Odijk, Jan H.J. Hoeijmakers, Roland Kanaar,

RNA Interference and Gene Delivery

Article15 November 2001free access The structure-specific endonuclease Ercc1—Xpf is required for targeted gene replacement in embryonic stem cells Laura J. Niedernhofer Laura J. Niedernhofer Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Jeroen Essers Jeroen Essers Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Geert Weeda Geert Weeda Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Berna Beverloo Berna Beverloo Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Jan de Wit Jan de Wit Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Manja Muijtjens Manja Muijtjens Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Hanny Odijk Hanny Odijk Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Jan H. J. Hoeijmakers Jan H. J. Hoeijmakers Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Roland Kanaar Corresponding Author Roland Kanaar Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Department of Radiation Oncology, University Hospital, Rotterdam/Daniel, The Netherlands Search for more papers by this author Laura J. Niedernhofer Laura J. Niedernhofer Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Jeroen Essers Jeroen Essers Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Geert Weeda Geert Weeda Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Berna Beverloo Berna Beverloo Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Jan de Wit Jan de Wit Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Manja Muijtjens Manja Muijtjens Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Hanny Odijk Hanny Odijk Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Jan H. J. Hoeijmakers Jan H. J. Hoeijmakers Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Roland Kanaar Corresponding Author Roland Kanaar Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands Department of Radiation Oncology, University Hospital, Rotterdam/Daniel, The Netherlands Search for more papers by this author Author Information Laura J. Niedernhofer1, Jeroen Essers1, Geert Weeda1, Berna Beverloo1, Jan de Wit1, Manja Muijtjens1, Hanny Odijk1, Jan H. J. Hoeijmakers1 and Roland Kanaar 1,2 1Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands 2Department of Radiation Oncology, University Hospital, Rotterdam/Daniel, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6540-6549https://doi.org/10.1093/emboj/20.22.6540 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Ercc1—Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1—Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1—Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1—Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1—Xpf in making the recipient genomic locus receptive for gene replacement. Introduction hERCC1 was originally identified as the human gene that corrects hypersensitivity to UV irradiation in one of 11 Chinese hamster ovary (CHO) cell complementation groups (Westerveld et al., 1984), hence the name excision repair cross complementation group 1. The gene is highly conserved with homologs in mouse, mErcc1 (Selfridge et al., 1992), Saccharomyces cerevisiae, RAD10 (van Duin et al., 1986) and Schizosaccharomyces pombe, swi10 (Rodel et al., 1992). In cells, the Ercc1 protein exists in a tight complex with Xpf (Biggerstaff et al., 1993; van Vuuren et al., 1993). Heterodimerization is required to stabilize both proteins and for complementation of DNA repair defects. Xpf is also highly conserved (Sijbers et al., 1996), as is heterodimeric interaction. The Rad10—Rad1 protein complex was discovered to be an endonuclease (Tomkinson et al., 1993) that cleaves double-stranded (ds) DNA only on the strand with a 3′ single-strand (ss) tail (Bardwell et al., 1994). This substrate specificity has helped to define the role of Ercc1—Xpf in correcting UV sensitivity. The complex is responsible for making a cut in the damaged strand of duplex DNA 5′ to a lesion in nucleotide excision repair (NER) (Mu et al., 1995; Sijbers et al., 1996). In addition, the complex cleaves stem—loop and splayed arm structures within duplex DNA and only on the strand with the 3′ ss tail (de Laat et al., 1998a). Although ERCC1 plays an essential role in NER, it is unable to complement the UV sensitivity of any of the eight complementation groups of fibroblast cell lines derived from NER-deficient xeroderma pigmentosum patients (van Duin et al., 1989). Nor has a viable mutation in hERCC1 been described. Furthermore, Ercc1−/− mice display a much more severe phenotype than other NER-deficient mouse models (McWhir et al., 1993; Weeda et al., 1997). These observations imply additional function(s) for Ercc1 outside NER. Hints as to this additional function(s) come from studies of mutant cell lines. Ercc1- and Xpf-deficient mammalian cells, unlike other NER-deficient cells, are sensitive to DNA crosslinking agents (Westerveld et al., 1984; Hoy et al., 1985). However, they are not ionizing radiation (IR)-sensitive. This is also true for rad10, rad1 (Moore, 1978) and swi10 mutants (Hang et al., 1996). These observations indicate a role for Ercc1—Xpf in NER-independent interstrand crosslink repair but not in double-strand break (DSB) repair. In addition to involvement in NER and interstrand crosslink repair, there is evidence for a role of Ercc1—Xpf in recombination. For example in S.cerevisiae, mutations in rad1 or rad10 suppress mitotic recombination between direct or inverted repeats (Klein, 1988; Schiestl and Prakash, 1988, 1990; Prado and Aguilera, 1995). Furthermore, recombination between repeats yields different products in rad1 and rad10 compared with wild-type (wt) strains (Klein, 1988; Aguilera and Klein, 1989; Zehfus et al., 1990). Excisional products in which the sequence between the repeats is lost are less frequently recovered from the mutant strains. Similar results were obtained in Ercc1-deficient CHO cells (Sargent et al., 2000). The rad1 phenotype is not exaggerated by a mutation in rad10 (Schiestl and Prakash, 1990), implying that the role these proteins play in recombination is dependent upon protein heterodimer formation and therefore most likely its endonuclease function. Intrachromosomal recombination between directly repeated sequences occurs most efficiently via single-strand annealing (SSA) (Paques and Haber, 1999). SSA requires a DSB between or within one of the repeats followed by exonuclease resection from both ends revealing complementary 3′ ss overhangs. The regions of homology within the two overhangs align and anneal, ultimately yielding a product in which one of the repeats and all intervening sequences are lost. The proposed role for Ercc1—Xpf in SSA is to remove nonhomologous termini from the 3′ ss overhangs after the overhangs have realigned. Indeed, Rad1 and Rad10 do cleave nonhomologous ss termini from the ends of a DSB to facilitate repair via homologous recombination (HR) in yeast (Fishman-Lobell and Haber, 1992; Ivanov and Haber, 1995; Prado and Aguilera, 1995; Paques and Haber, 1997), as do the hamster homologs in CHO cells (Sargent et al., 2000). Similarly, Ercc1 is required to remove nonhomologous tails from ends-in targeting constructs in CHO cells (Adair et al., 2000). There is also mounting evidence that Ercc1—Xpf (and their homologs) participate in the processing of recombination intermediates in which the DNA substrates are not identical, but the sequence nonhomology is positioned between homologous domains rather than at an end. For example, gene conversion products resulting from recombination between direct repeats that differ by distally positioned deletions are not recovered in a rad1 strain (Klein, 1988). Similarly, in rad1 and rad10 strains, gene targeting using a linear construct is suppressed 40-fold (Schiestl and Prakash, 1988, 1990; Saparbaev et al., 1996). Furthermore, mutations in rad1 or rad10 suppress meiotic repair of loops created by patches of nonhomology between homologous chromosomes (Kirkpatrick and Petes, 1997; Kearney et al., 2001) as do mutations in the S.pombe homologs (Fleck et al., 1999). Finally, in Ercc1-deficient CHO cells intrachromosomal recombination between direct repeats separated by 600 bp of intervening sequence yields many more gene rearrangements compared with Ercc1-proficient cells (Sargent et al., 1997), in particular deletions beginning at the borders between the repeat and the intervening sequence. In the experiments described below, we discovered that Ercc1 is absolutely required for HR between gene replacement constructs and genomic loci in mouse embryonic stem (ES) cells. We tested linear constructs consisting of a selectable marker flanked by completely homologous arms and constructs including additional nonhomologous termini. This allowed us to determine whether the essential function of Ercc1 in gene replacement was removal of 3′ nonhomologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Our data are consistent with the latter and have implications for the mechanism of targeted gene replacement in mammalian cells. Results Generation of mErcc1−/− and rescued ES cells A clonal mErcc1+/− [neomycin-resistant (neoR)] ES cell line (Weeda et al., 1997) was transfected with a linearized mErcc1 targeting construct containing a hygromycin selectable marker (hygroR) with the goal of disrupting the second mErcc1 allele and thereby generating a nullizygous cell line (Figure 1A). More than 300 hygroR clones from multiple transfection experiments were analyzed by DNA blot analysis (Figure 1B). The absolute targeting efficiency of the mErcc1 locus was 16% as previously reported (Weeda et al., 1997). However, there was disequilibrium between targeting the wt and mutant alleles of the mErcc1+/− cells. In only two out of 48 targeted clones was the wt mErcc1 allele replaced by the hygroR construct. Thus, the frequency of generating mErcc1−/− cells was 12-fold lower than expected, suggesting selection against Ercc1-deficient ES cells. Indeed, the mErcc1−/− cells required daily passaging to maintain morphology and growth characteristics typical of wt ES cells. Figure 1.Targeting strategy for sequential inactivation of both mErcc1 alleles in mouse ES cells. (A) The top of the scheme depicts the 3′ end of the mErcc1 genomic locus with relevant restriction sites. Numbered boxes indicate exons. Beneath, the two targeting constructs are depicted. Exon 7 of mErcc1 was disrupted by insertion of either a neoR or hygroR marker containing a diagnostic BamHI restriction site. ES cells were transfected first with the mErcc1neoR construct. Genomic DNA was isolated from resistant clones, digested with BamHI and analyzed by DNA hybridization using a 5′ external probe (indicated above the constructs). The lengths of the expected genomic fragments are indicated with horizontal arrows below the constructs. mErcc1+/− cells were subsequently transfected with the mErcc1hygroR construct and grown under hygromycin B selection. Positive clones were analyzed by DNA blotting. (B) An example of a DNA blot of BamHI-digested genomic DNA isolated from ES clones and probed with the 5′ external probe is shown. The probe detects either an 8-kb fragment (wt allele), 4.8-kb fragment (hygroR mutant) or 4.2-kb allele (neoR mutant). Two independent double mutant clones were identified. Download figure Download PowerPoint To verify that the phenotype of the mErcc1−/− cells was directly attributable to the loss of functional Ercc1, we generated rescued mutant lines. mErcc1−/− ES cells were transfected with a plasmid harboring the hERCC1 cDNA fused in-frame with a downstream sequence encoding yellow fluorescent protein (YFP). Transformants were screened for expression of ERCC1—YFP by fluorescence microscopy. In all of the clones screened, the fluorescent signal was low and restricted to the nucleus. These characteristics are inconsistent with expression of YFP alone. Immunoblot analysis of whole cell extracts (WCE) from the individual clones with α-hERCC1 antibody indicated expression of full-length ERCC1—YFP fusion protein but not ERCC1 by itself (Figure 2A). Furthermore, fusion protein levels were similar to levels of ERCC1 in HeLa cells, indicating physiological expression levels. The α-hERCC1 antibody does not recognize mouse Ercc1, therefore the absence of a protein signal in mErcc1−/− cell extract does not formally prove that there is no endogenous Ercc1 present in the mutant cell lines. However, immunoblot analysis with α-hXPF antibody, which does recognize the endogenous mouse protein, demonstrates markedly reduced levels of Xpf protein in the mErcc1−/− lines (Figure 2B). This is consistent with previous findings of destabilization of Xpf in the absence of Ercc1 (Sijbers et al., 1996; Houtsmuller et al., 1999; Gaillard and Wood, 2001). Xpf protein levels in the rescued mutant line were comparable to wt ES cells. Figure 2.Immunoblot analysis of wt, mErcc1−/− and rescued mutant ES cell lysates. (A) Immunoblot of WCE using α-hERCC1 polyclonal antibody. The antibody recognizes a 39 kDa band in the positive control (HeLa cells, lane 6). Lanes 2–4 contain lysates from three cell lines (#8, 9 and 10) expanded from individual MMC-resistant clones established by stable transfection of the mErcc1−/− ES cells with a construct containing the hERCC1 cDNA coupled in-frame with YFP. Expression of only the full-length fusion protein is seen as a single band with the predicted molecular mass of 66 kDa (39 kDa ERCC1 + 27 kDa YFP). Lane 5 contains a lysate from mErcc1−/− ES cells. (B) Immunoblot of WCE using α-hXPF polyclonal antibody. A band corresponding to XPF (115 kDa) is visible in the HeLa cell and wt ES cell lysates (lanes 2 and 3, respectively) but not in the mErcc1−/− mutant line (lane 4). Rescue of the mutant line with hERCC1—YFP restores the Xpf band to physiological levels (lane 5). The presence of a cross-reactive band in the ES cells (∼75 kDa) serves as a loading control. Download figure Download PowerPoint Characterization of mErcc1−/− ES cells To characterize the mErcc1−/− cells, we examined their sensitivity to three types of DNA damaging agents: UV, mitomycin C (MMC) and IR. UV causes lesions that are substrates for NER, while MMC induces interstrand crosslinks and IR generates DSBs that in ES cells are efficiently repaired by HR (Dronkert et al., 2000). We observed that the mErcc1−/− cells are 2.7-fold more sensitive to UV than wt cells (Figure 3A). In fact, the Ercc1 mutant was equally as sensitive to UV irradiation as an Xpa−/− ES line (H.de Waard, unpublished data) consistent with a complete NER defect. The cell lines stably transfected with hERCC1 cDNA behaved identically to the parental wt line, demonstrating a complete restoration of NER. The mErcc1−/− cells were 10-fold more sensitive to low doses of MMC than the wt and rescued mutant cells (Figure 3B). These results were not crosslink agent-specific, as the mErcc1−/− ES cells were also 16-fold more sensitive to the crosslinking agent cisplatin and 12-fold more sensitive to 8-methoxypsoralen + UVA than the wt cells (data not shown). The mErcc1−/− cells were not sensitive to IR (Figure 3C). This was in contrast to the HR-defective mRad54−/− ES. Finally, to screen further for a role for Ercc1 in HR, the frequency of sister chromatid exchange (SCE) was measured in the different ES cell lines. The mErcc1−/− cells displayed the same number of SCEs as the parental wt line or the rescued mutant lines (data not shown), indicating that the absence of functional Ercc1 does not preclude HR. This was true whether spontaneous or MMC-induced SCEs were measured. Together, these data define the mErcc1−/− ES cells as NER-deficient, crosslink repair-deficient but DSB repair- and HR-proficient. Figure 3.Effect of UV, MMC and IR on wt and mErcc1−/− ES cells. (A) Clonogenic survival of wt (squares), mErcc1−/− (triangles) and a rescued mutant line (circles) after UV irradiation. The percent of irradiated cells that were able to form a colony compared with untreated cells is plotted against the dose of UV. (B) Clonogenic survival of wt, mErcc1−/− and a rescued mutant line after treatment with MMC for 1 h. (C) Clonogenic survival of ES cells after treatment with IR. Since the mErcc1−/− line was not hypersensitive to irradiation, a rescued line was not tested. An mRad54−/− ES line (diamonds) was used as a positive control for irradiation. Download figure Download PowerPoint Measuring gene replacement frequency in mErcc1−/− ES cells To explore the possibility of a role for Ercc1 in recombination between non-identical substrates, we examined the capacity of the mutant cells for gene targeting. In order to do this, the mutant, wt and rescued ES cells were transfected with a linear targeting construct consisting of a puromycin resistance (puroR) selectable marker flanked on both sides with ∼4 kb of sequence homologous to the mRad54 genomic locus (Figure 4). The construct was isolated either with or without 14–32 bp of heterologous sequences at each end. Figure 4.Strategy for quantitating gene replacement in wt and mErcc1−/− ES cells. (A) Top, the central portion of the mRad54 genomic locus is shown. Exons are depicted as numbered boxes. The targeting construct was generated by inserting a puroR expression cassette into intron 3. Once homologously recombined into the genome, the StuI fragment encompassing this region is shortened from 9.0 to 8.5 kb. The targeting construct was isolated from a vector as either an EcoRI fragment (completely homologous to the mRad54 locus, with the exception of the selectable marker), or a ClaI—NotI fragment (which contains 32 bp of bacterial sequence on the 5′ and 14 bp on the 3′ end of the construct, indicated as dashed lines; not drawn to scale). Stable transformants were selected with puromycin. (B) Genomic DNA was isolated from individual resistant clones and analyzed by DNA hybridization after StuI digestion. Targeting was detected by the appearance of an 8.5-kb DNA fragment using a 3′ external probe from exons 7 and 8 of mRad54 cDNA. Download figure Download PowerPoint In the wt cell line, the construct with the nonhomologous termini targeted the mRad54 locus with 30% efficiency (Table I). In stark contrast, transfection of the same construct into mErcc1−/− cells did not yield a single targeting event in >225 puroR clones. This represents a >78-fold drop in targeting efficiency. In the mErcc1−/− ES line rescued with a tagged hERCC1 cDNA, the targeting efficiency returned to wt levels. Unexpectedly, the complete absence of targeting events in the mErcc1−/− ES cells was also observed when the ends of the transfected linear construct were completely homologous to the genomic locus. Thus, these data define a new role for Ercc1 in gene targeting in mammalian cells that is distinct from the previously identified role in removing nonhomologous termini from recombinational intermediates. Table 1. Frequency of gene replacement events in ES cells of the indicated genotypes ES cell genotype Targeted locus: mRad54 mRad54 mRb mCsb Construct ends: non-homologous homologous non-homologous non-homologous wt 30.9%a (30/97) 25.7% (37/144) 20.0%e,f (19/95) 17.9%e (10/56) mErcc1−/− <0.4%b (0/227) <0.4%b (0/269) <0.5%b (0/212) <0.6%b (0/160) mErcc1−/− + hERCC1 14.7%c,d (21/143) 26.1%c,d (36/138) n.d. n.d. mRad54−/− 1.6% (4/249) 2.1% (6/284) 3.3%e (2/61) 4.3%e (3/69) n.d., not determined. a Percent of homologous integration events amongst the total number of integration events. Absolute numbers are shown in parentheses. b The difference between the mErcc1−/− ES cells and the wt cells is statistically significant for all targeting constructs (p 0.01). The difference between the rescued mErcc1−/− cell line and the mErcc1−/− ES cells is significant (p 0.05). e Previously reported (Essers et al., 1997). f This data was collected in a mRad54+/− ES cell line (Essers et al., 1997). To exclude the possibility that the abrogation of gene targeting in mErcc1−/− ES cells was construct related, we also tested other linear 'ends-out' constructs that target the murine retinoblastoma (mRb) or Cockayne syndrome B (mCsb) locus (Table I). With these additional constructs, we again never observed a single event of gene replacement (0/212 puroR clones and 0/160 puroR clones, respectively) in the Ercc1-deficient cells, although the targeting efficiency was 18–20% in wt ES cells. Nor was the abrogation of gene targeting peculiar to the particular Ercc1−/− cell line studied, as examination of a second Ercc1−/− and two additional rescued mutant lines yielded results in accordance with Table I (0/165 puroR clones in the mutant cell line; 7/38 and 6/30 puroR clones in the rescued line). By way of comparison, we also determined the targeting frequency of these same constructs in an HR-deficient mRad54−/− ES cell line. In the mRad54−/− cells, targeted integration was reduced by 5- to 15-fold, but not abolished. The difference in the level of suppression of gene replacement between the mErcc1−/− and mRad54−/− ES cells was statistically significant (p 200-fold drop in targeting efficiency in the Ercc1-deficient cells compared with wt ES cells. Stable transfection of the Ercc1-deficient cells with a tagged hERCC1 cDNA corrects the targeting efficiencies to wt levels. Surprisingly, failure to homologously recombine targeting constructs in the mErcc1−/− cells is not limited to constructs carrying heterologous terminal sequences. This provides the first evidence that in mammalian cells, Ercc1 is required for HR between non-identical DNA substrates in a capacity other than removing nonhomologous termini. The data obtained with the mErcc1−/− ES cells can be contrasted with the results obtained with mRad54−/− ES cells. Rad54-deficient cells are sensitive to IR (Figure 3 and Essers et al., 1997) and have a reduced capacity for spontaneous and DNA damage-induced SCE (Dronkert et al., 2000), yet gene replacement is not abrogated in mRad54−/− ES cells (Table I and Essers et al., 1997). This serves to highlight the differential roles of the proteins in HR; whereas Rad54 facilitates the general mechanism of HR, Ercc1 has a limited, yet essential function in a subset of recombinational events. The finding that Ercc1 plays a role in gene replacement in ES cells is consistent with observations made in S.cerevisiae, the first organism for which targeted gene replacement was developed (Orr-Weaver et al., 1981; Rothstein, 1983). Replacement is suppressed 40-fold in rad1 or rad10 deletion strains compared with wt strains (Schiestl and Prakash, 1988, 1990; Saparbaev et al., 1996). In contrast, however, the role of Ercc1 in mammalian cells appea