Cells from ercc1-deficient mice show increased genome instability and a reduced frequency of s-phase-dependent illegitimate chromosome exchange but a normal frequency of homologous recombination

Abstract

The ERCC1 protein is essential for nucleotide excision repair in mammalian cells and is also believed to be involved in mitotic recombination. ERCC1-deficient mice, with their extreme runting and polyploid hepatocyte nuclei, have a phenotype that is more reminiscent of a cell cycle arrest/premature ageing disorder than the classic DNA repair deficiency disease, xeroderma pigmentosum. To understand the role of ERCC1 and the link between ERCC1-deficiency and cell cycle arrest, we have studied primary and immortalised embryonic fibroblast cultures from ERCC1-deficient mice and a Chinese hamster ovary ERCC1 mutant cell line. Mutant cells from both species showed the expected nucleotide excision repair deficiency, but the mouse mutant was only moderately sensitive to mitomycin C, indicating that ERCC1 is not essential for the recombination-mediated repair of interstrand cross links in the mouse. Mutant cells from both species had a high mutation frequency and the level of genomic instability was elevated in ERCC1-deficient mouse cells, both in vivo and in vitro. There was no evidence for an homologous recombination deficit in ERCC1 mutant cells from either species. However, the frequency of S-phase-dependent illegitimate chromatid exchange, induced by ultra violet light, was dramatically reduced in both mutants. In rodent cells the G1 arrest induced by ultra violet light is less extensive than in human cells, with the result that replication proceeds on an incompletely repaired template. Illegitimate recombination, resulting in a high frequency of chromatid exchange, is a response adopted by rodent cells to prevent the accumulation of DNA double strand breaks adjacent to unrepaired lesion sites on replicating DNA and allow replication to proceed. Our results indicate an additional role for ERCC1 in this process and we propose the following model to explain the growth arrest and early senescence seen in ERCC1-deficient mice. In the absence of ERCC1, spontaneously occurring DNA lesions accumulate and the failure of the illegitimate recombination process leads to the accumulation of double strand breaks following replication. This triggers the p53 response and the G2 cell cycle arrest, mediated by increased expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1). The increased levels of unrepaired lesions and double strand breaks lead to an increased mutation frequency and genome instability.

Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination

“…These results suggest that ERCC1 is not generally required for targeted homologous recombination and demonstrate that the short terminal non‐homologies blocking both arms of targeting homology in the pAG6 vector can be effectively removed by ERCC1 ‐independent pathways. While our results appear to support the conclusion of Melton et al . (1998) that ERCC1 is not required for the general recombination pathways involved in gene targeting, they are difficult to reconcile with findings by Schiestl and Prakash (1988, 1990) that in yeast, RAD1 or RAD10 knockouts markedly reduce the frequencies of targeted integration by linearized insertion vectors with no terminal non‐homology.…”

“…In S.cerevisiae , the Rad1/Rad10 endonuclease appears to be required not only for removal of long, 3′‐ended, non‐homologous single‐stranded tails from SSA recombination intermediates during DSB‐induced recombination between direct repeats, but also for the removal of such blocking non‐homologies from the 3′‐OH ends of invading homologous strands in other pathways of DSB‐induced homologous recombination (Fishman‐Lobell and Haber, 1992; Ivanov and Haber, 1995; Prado and Aquilera, 1995; Saparbaev et al ., 1996; Sugawara et al ., 1997; Paques and Haber, 1997, 1999; Colaiacovo et al ., 1999). Although two previous studies in mammalian cells failed to demonstrate a general effect of ERCC1 deficiency on either the rate of spontaneous intrachromosomal homologous recombination between direct repeats (Sargent et al ., 1997) or the overall frequency of gene targeting (Melton et al ., 1998), neither study was designed to investigate the role of ERCC1 in recombination events that would specifically require the removal of terminal non‐homologies. To examine this most likely role of ERCC1 directly, we conducted gene targeting experiments employing two insertion‐type vectors (pAG6 and pAG6ins0.9), in which both arms of APRT targeting homology were blocked by very short or substantially longer terminal non‐homologies flanking each side of a DSB.…”

“…The inability of ERCC1 − cells to remove long terminal non‐homologies from the 3′‐OH ends of DNA strands during SSA‐ or SDSA‐mediated recombination between chromosomal repeat sequences could lead to DSBs during DNA replication. These DSBs could in turn promote end‐joining by non‐homologous/illegitimate recombination pathways, leading to increased frequencies of deleterious deletions or chromosomal rearrangements that might contribute to the genomic instability observed in ERCC1 − knockout mice (McWhir et al ., 1993; Weeda et al ., 1997; Melton et al ., 1998).…”

“…In studies examining the potential role of ERCC1 in recombination in mammalian cells, no substantial differences were observed between ERCC1 + and ERCC1 − cells in overall frequencies of homologous recombination between extrachromosomal substrates (Nairn et al ., 1991), plasmid‐chromosome targeting in ERCC1 nullizygous mouse embryonic stem cells (Melton et al ., 1998), or intrachromosomal recombination between direct repeats in CHO ERCC1 + and ERCC1 − cell lines (Sargent et al ., 1997). However, cells from ERCC1 knockout mice show increased genomic instability (Melton et al ., 1998) and recombination‐dependent deletions and rearrangements involving duplicated sequences at the Chinese hamster ovary (CHO) APRT locus appear to be suppressed in ERCC1 + cells relative to ERCC1 − cells (Sargent et al ., 1997). These phenotypes are suggestive of a subtle, rather than general, recombination defect in ERCC1 − cells.…”

No Correlation between the Expression of FXR and Genes Involved in Multidrug Resistance Phenotype of Primary Liver Tumors

“…Nevertheless, the importance of MOC-IV may change during pharmacological treatment. Several studies have shown that chronic treatment with cisplatin could induce the expression of many MOC-IV genes. − Thus, in the present study HepG2 cells were found to be very sensitive to treatment with cisplatin, which induced a marked and generalized increase in the expression of the genes included in MOC-IV.…”