Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1.
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 RADIO 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.
Sargent
R G
RG
The Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA.
Rolig
R L
RL
Kilburn
A E
AE
Adair
G M
GM
Wilson
J H
JH
Nairn
R S
RS
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004720:Endonucleases; D011506:Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D013937:Thymidine Kinase / Q000235:genetics
D004268:DNA-Binding Proteins; D011506:Proteins; D000228:Adenine Phosphoribosyltransferase; D013937:Thymidine Kinase; D004720:Endonucleases