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The study of gene function has been greatly facilitated by the development of strategies to modify genomic DNA. Gene targeting is one of the most successfully applied techniques used to examine the roles of specific genes in a wide variety of model systems from yeast to mammals. Our laboratory has pioneered the use of the Chinese hamster ovary (CHO) cell culture model system to study pathways of DNA repair and recombination at the hemizygous CHO APRT locus. By using a simple and effective gene targeting method, we have generated a number of DNA repair-deficient cell lines that have been used in targeted recombination experiments to investigate pathways of recombinational repair in somatic mammalian cells. These methods can be readily customized to generate a variety of cell lines deficient in specific genes of interest and can be applied to study the roles of other DNA repair proteins in pathways of mammalian recombinational repair.

The ERCC1-XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1-XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10-Rad1 in budding yeast that ERCC1-XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study., (Copyright © 2010 Elsevier B.V. All rights reserved.)

DNA interstrand crosslinks (ICLs) are among the most deleterious cytotoxic lesions encountered by cells, mainly due to the covalent linkage these lesions create between the two strands of DNA which effectively blocks replication and transcription. Although ICL repair in mammalian cells is not fully understood, processing of these lesions is thought to begin by "unhooking" at the site of the damaged base accompanied by the generation of a double strand break and ultimately repair through translesion synthesis and homologous recombination. A key player in this repair process is the heterodimeric protein complex ERCC1-XPF. Although some models of ICL repair restrict ERCC1-XPF activity to the unhooking step, recent data suggest that this protein complex acts in additional downstream steps. Here, we review the evidence implicating ERCC1-XPF in multiple steps of ICL repair., (Environ. Mol. Mutagen., 2010. (c) 2010 Wiley-Liss, Inc.)

Gene targeting by homologous recombination in mammalian cells is an important tool for generating genetically modified mice used for modeling human diseases. Gene targeting approaches are also useful for studying the mechanisms of homologous recombination. We have developed gene targeting methods that we have specifically used to investigate the mechanisms of recombination in cultured mammalian cells. In this chapter, we describe the generation of Chinese hamster ovary (CHO) cell gene disruption ("knockout") mutants in the repair/recombination gene ERCC1. Using this approach, we have constructed pairs of isogenic ERCC1-proficient and -deficient (null) CHO cell lines and used them as recipients for gene targeting assays in which a hemizygous mutant hamster adenine phosphoribosyltransferase (APRT) locus is corrected by homologous recombination with plasmid vectors containing hamster APRT DNA sequence homologous to the target gene in each cell line. The configuration of the targeting vector leads to experimental outcomes in which certain classes of APRTrecombinants are over- or under-represented depending on the repair gene status of the transfection recipient. We describe methods both for targeted gene knockout of ERCC1, and for APRT targeted gene correction by homologous recombination, and some of our experimental results using these approaches.

DNA damage is preferentially repaired in the transcribed strand of many active genes. Although the concept of DNA repair coupled with transcription has been widely accepted, its mechanisms remain elusive. We recently reported that in Chinese hamster ovary cells while ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in the transcribed strand of dihydrofolate reductase gene, CPDs are efficiently repaired in both strands of adenine phosphoribosyltransferase (APRT) locus, in either a transcribed or nontranscribed APRT gene (1). These results suggested that the transcription dependence of repair may depend on genomic context. To test this hypothesis, we constructed transfectant cell lines containing a single, actively transcribed APRT gene, integrated at different genomic sites. Mapping of CPD repair in the integrated APRT genes in three transfectant cell lines revealed two distinct repair patterns, either preferential repair of CPDs in the transcribed strand or very poor repair in both strands. Similar kinetics of micrococcal nuclease digestion were seen for all three transfectant APRT gene domains and endogenous APRT locus. Our results suggest that both the efficiency and strand-specificity of repair of an actively transcribed gene are profoundly affected by genomic context but do not reflect changes in first order nucleosomal structure.

The XpF/Ercc1 structure‐specific 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 non‐homologies flanking 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‐homologies—almost complete ablation of gap repair and single‐reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1+ cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non‐homologous tails from invading homologous strands. [ABSTRACT FROM AUTHOR]

Abstract: The UV hypersensitive CHO cell mutant UV41 is the archetypal XPF mammalian cell mutant, and was essential for cloning the human nucleotide excision repair (NER) gene XPF by DNA transfection and rescue. The ERCC1 and XPF genes encode proteins that form the heterodimer responsible for making incisions required in NER and the processing of certain types of recombination intermediates. In this study, we cloned and sequenced the CHO cell XPF cDNA, determining that the XPF mutation in UV41 is a +1 insertion in exon 8 generating a premature stop codon at amino acid position 499; however, the second allele of XPF is apparently unaltered in UV41, resulting in XPF heterozygosity. XPF expression was found to be several-fold lower in UV41 compared to its parental cell line, AA8. Using approaches we previously developed to study intrachromosomal recombination in CHO cells, we modified UV41 and its parental cell line AA8 to allow site-specific gene targeting at a Flp recombination target (FRT) in intron 3 of the endogenous adenine phosphoribosyltransferase (APRT) locus. Using FLP/FRT targeting, we integrated a plasmid containing an I-SceI endonuclease sequence into this site in the paired cell lines to generate a heteroallelic APRT duplication. Frequencies of intrachromosomal recombination between APRT heteroalleles and the structures of resulting recombinants were analyzed after I-SceI induction of site-specific double-strand breaks (DSBs) in a non-homologous insertion contained within APRT homology. Our results show that I-SceI induced a small proportion of aberrant recombinants reflecting DSB-induced deletions/rearrangements in parental, repair-proficient AA8 cells. However, in XPF mutant UV41, XPF heterozygosity is responsible for a similar, but much more pronounced genomic instability phenotype, manifested independently of DSB induction. In addition, gene conversions were suppressed in UV41 cells compared to wild-type cells. These observations suggest that UV41 exhibits a genomic instability phenotype of aberrant recombinational repair, confirming a critical role for XPF in mammalian cell recombination. [Copyright &y& Elsevier]