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Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1 - PubMed

  • ️Wed Jan 01 1997

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

R G Sargent et al. Proc Natl Acad Sci U S A. 1997.

Abstract

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.

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Figures

Figure 1
Figure 1

Gene structures of the APRT locus and a possible recombination intermediate recognized by NER. (A) Tandemly duplicated APRT recombination substrate. In this and other figures, the heavy lines represent chromosomal sequences and the thin line represents plasmid backbone. The APRT gene is represented by an open box, the GPT gene by a solid box, and the TK gene by a crosshatched box. The FRT sequences located in intron 2 are represented by the inverted triangle above both APRT gene copies. The exon 2 mutation that destroys the EcoRV site is denoted by a heavy vertical line at the 5′ end of the upstream APRT copy; at other positions (see Table 2) the heavy line represents undefined APRT point mutations. The sizes of the three recombination intervals defined by the TK gene, GPT gene, and exon-2 mutation are shown above the APRT map. (B) Single copy APRT gene in cell lines used for measuring spontaneous rates of point mutations, deletions, and gene rearrangements.

Figure 2
Figure 2

Molecular structure of APRT− gene deletions and rearrangements recovered from ERCC1− cells. APRT exons are indicated as open boxes. Numbering in kilobase pairs (kb) for these maps is relative to the BamHI site 5′ of the downstream APRT copy (map position 0). The PCR primers used to map the extent of deletions and rearrangements are shown below the APRT maps. The open areas between the brackets for each gene structure represent regions that did not yield PCR products; thus the bracketed regions indicate the interval in which deletion and rearrangement junctions map. The sizes of the deletions as estimated from Southern analysis are indicated at the right. Deletions whose endpoints were determined precisely by sequencing across PCR products are indicated without brackets. A straight line under GPT for both tandem duplication and crossover recombinant structures indicates GPT is present (by PCR); an indentation under the GPT gene indicates it is absent, which may indicate conversion to the wild-type APRT sequence. Rearrangement 1 is missing the GPT gene, perhaps because of gene conversion, but it also has other rearrangements that are apparent from Southern analysis.

Figure 3
Figure 3

A recombination heteroduplex intermediate that could be a substrate for the Ercc1/XpF endonuclease. The heteroduplex DNA was created by annealing the top DNA strand from the upstream APRT gene to the bottom strand from the downstream APRT gene so that the heteroduplex spans the GPT gene (see Fig. 1). This heteroduplex creates an ≈800-bp deletion loop similar to in vitro substrates processed by purified NER proteins, whose putative sites of action are indicated.

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