Disruption of Mouse SNM1 Causes Increased Sensitivity to the DNA Interstrand Cross-Linking Agent Mitomycin C

Abstract

DNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to nucleotide excision, recombination, and translesion DNA repair pathways in Escherichia coli, Saccharomyces cerevisiae, and mammals. We cloned and analyzed mammalian homologs of the S. cerevisiae gene SNM1 (PSO2), which is specifically involved in ICL repair. Human Snm1, a nuclear protein, was ubiquitously expressed at a very low level. We generated mouse SNM1 ؊/؊ embryonic stem cells and showed that these cells were sensitive to mitomycin C. In contrast to S. cerevisiae snm1 mutants, they were not significantly sensitive to other ICL agents, probably due to redundancy in mammalian ICL repair and the existence of other SNM1 homologs. The sensitivity to mitomycin C was complemented by transfection of the human SNM1 cDNA and by targeting of a genomic cDNA-murine SNM1 fusion construct to the disrupted locus. We also generated mice deficient for murine SNM1. They were viable and fertile and showed no major abnormalities. However, they were sensitive to mitomycin C. The ICL sensitivity of the mammalian SNM1 mutant suggests that SNM1 function and, by implication, ICL repair are at least partially conserved between S. cerevisiae and mammals.

Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation

“…A knockout of the murine SNM1 gene renders ES cells moderately sensitive to ICL induced by MMC. However, this effect appears to be somewhat specific to MMC, as sensitivity to other crosslinking agents, such as cisplatin and melphalan, is not increased in these knockouts (Dronkert et al, 2000). In contrast, hSNM1C/ARTEMIS functions in the pathway for DSB repair.…”

“…No proteins migrating at the predicted size for hSNM1B were detected in immunoblots from HeLa cell lysates or immunoprecipitates, despite the presence of hSNM1B transcripts in these cells detectable by RT-PCR. Interestingly, the yeast homolog PSO2, the human and mouse SNM1 and the human SNM1C/ARTEMIS are all expressed at very low levels, and the failure to detect hSNM1B raises the possibility that this is a common feature of proteins of the SNM1B family (Richter et al, 1992;Dronkert et al, 2000;Moshous et al, 2001).…”

“…A genomic clone providing a partial sequence of hSNM1B was previously identified based on its sequence similarity to yeast PSO2 and human SNM1 (Dronkert et al, 2000). We used this partial sequence as a template in an NCBI nucleotide database search and identified numerous cDNA clones with a perfect match.…”

“…Four additional sequences with similarity to the yeast PSO2 gene have been reported: hSNM1C, also known as ARTEMIS, a partial sequence of hSNM1B (Dronkert et al, 2000), and the more distantly related ELAC2 (Tavtigian et al, 2001) and CPSF73 (Jenny et al, 1996). The sequence similarity between the PSO2 gene and these mammalian proteins derives largely from a shared metallo-b-lactamase domain, which is predicted to play a role in DNA and RNA metabolism (Ma et al, 2002).…”

“…Mouse embryonic stem (ES) cells with disrupted mSNM1 display a twofold increase in their sensitivity to MMC, but not to other DNA crosslinking agents (Dronkert et al, 2000). Richie et al (2002) characterized the subcellular distribution of hSNM1 using indirect immunofluorescence.…”