The XpF/Ercc1 structure-speci®c endonuclease performs the 5¢ incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/ Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/ Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1 + and ERCC1 ± cell lines with insertion vectors having long or short terminal nonhomologies¯anking each side of a double-strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT + recombinants recovered from ERCC1 ± cells using targeting vectors with long terminal non-homologiesÐ almost complete ablation of gap repair and singlereciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1 + cells. These results represent the ®rst demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, speci®cally in removal of long non-homologous tails from invading homologous strands. Keywords: ERCC1/gene targeting/homologous recombination/terminal non-homology/XpF±Ercc1 endonuclease IntroductionNucleotide excision repair (NER) is responsible for processing DNA lesions that cause large distortions in DNA helical structure, such as UV photoproducts and bulky covalent chemical adducts (de Laat et al., 1999). In eukaryotes, there is striking conservation of the genes involved in NER; the biochemical steps constituting this repair pathway are essentially the same in yeast (Saccharomyces cerevisiae) and mammalian cells (Aboussekhra and Wood, 1994;de Laat et al., 1999). After damage recognition, lesion demarcation and formation of a pre-incision complex, a dual incision step catalyzes release of a single-stranded oligonucleotide containing the damage site, allowing repair synthesis and ligation (Aboussekhra et al., 1995;Mu et al., 1996;de Laat et al., 1999). The proteins responsible for dual incision are structure-speci®c endonucleases that recognize transitional DNA duplex/single-stranded regions. In mammalian cells, the XpG protein performs the initial incision 2±9 nucleotides (nt) 3¢ to DNA damage and the XpF/Ercc1 complex makes the second incision 16±25 nt 5¢ to the damage (Matsunaga et al., 1996;Bessho et al., 1997b;Evans et al., 1997;de Laat et al., 1998). The precise location of the DNA incisions is dependent on the nature of the DNA lesion and results in excision of a 24±32 nt oligonucleotide fragment (Huang et al., 1992;Moggs et al., 1996).In S.cerevisiae, the RAD1 and RAD10 gene products also form a heterodimeric complex, which incises DNA speci®cally at 5¢-double-strand (ds)±3¢-single-strand (ss) junctions (Tomkinson et al., 1993;Bardwell et al., 1994) and the role of the Rad1/Rad10 endonuclease in NER in yeast is analogous to XpF/Ercc1 function in the d...

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1 ؊ and ERCC1 ؉ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT ؉ tandem duplication could give rise to APRT ؊ cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT ؊ products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1 ؊ cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1 ؊ cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1 ؊ cells are repaired by illegitimate recombination.

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese (Fig. la). A spontaneous 6-thioguanine-resistant, hypoxanthine (guanine) phosphoribosyltransferase (HPRT)-deficient subline, ATS-49tg, was used for targeting experiments.Cells were maintained as exponentially growing monolayer cultures in alpha modified Eagle's minimal essential medium (a-MEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin (20, 21).Plasmid DNAs. Plasmid pAG-7 (Fig. lb) 4574The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A summary is given for the lineage and complementation group assignments of 153 UV-sensitive mutants of the CHO AA8 cell line. The distribution of mutants among six complementation groups was highly non-random, with the great majority of the isolates belonging to groups 1 and 2. This asymmetry is consistent with the known hemizygosity of these two linked loci in CHO cells. The relative numbers of mutants induced in group 2 was found to depend greatly on the type of mutagen used. Mutagenesis with UV radiation, ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine and 7-bromomethylbenz[a]anthracene produced high frequencies of group 2 mutants. In contrast, ICR170 and ICR191, which are thought to produce mostly frameshift mutations, yielded very few mutants in group 2. These results are of particular importance in light of the recent finding that the human ERCC2 gene, which corrects group 2 mutants, has very strong homology with the yeast gene RAD3. RAD3 is an essential gene for viability in yeast, and the low recovery of group 2 mutants using the frameshift agents strongly suggests that frameshift mutations tend to be lethal in the hamster ERCC2 locus. Several mutagen-sensitive double mutants were isolated in two-step selections from EMS-, mitomycin C- or UV-sensitive parental cells, including the line UVU1, the first mammalian line with two mutations that affect UV sensitivity. The first mutation inactivated excision repair, and the second mutation appears to have affected some other recovery process. UVU1 should be useful for studying recovery processes that are separate from nucleotide excision repair.