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Disruption of mouse SNM1 causes increased sensitivity to the DNA interstrand cross-linking agent mitomycin C
M L Dronkert1, J de Wit, M Boeve
1Department of Cell Biology and Genetics, Centre for Biomedical Genetics, Erasmus University Rotterdam, The Netherlands.
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.
Insights
DNA interstrand cross-links (ICLs) cause lethal damage, but their repair is complex. Mammalian SNM1 is crucial for repairing ICLs, particularly sensitivity to mitomycin C, indicating conserved repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair Mechanisms
Background:
- DNA interstrand cross-links (ICLs) are severe DNA lesions that impede essential cellular processes like replication and transcription.
- Agents inducing ICLs are utilized in cancer chemotherapy due to their genotoxicity.
- ICL repair is a complex process involving multiple DNA repair pathways across different organisms.
Purpose of the Study:
- To investigate the role of mammalian homologs of the Saccharomyces cerevisiae SNM1 gene in DNA interstrand cross-link repair.
- To characterize the function of human SNM1 and generate mouse models deficient in SNM1 to assess its in vivo role.
Main Methods:
- Cloning and analysis of mammalian SNM1 homologs.
- Generation and characterization of SNM1-deficient mouse embryonic stem cells and mice.
- Assessing sensitivity to DNA interstrand cross-linking agents, including mitomycin C.
- Complementation studies using human SNM1 cDNA and a genomic fusion construct.
Main Results:
- Mammalian SNM1 was identified as a nuclear protein expressed at low levels.
- SNM1-deficient mouse embryonic stem cells and mice exhibited sensitivity to mitomycin C.
- Unlike yeast mutants, mammalian cells showed less sensitivity to other ICL agents, suggesting repair redundancy.
- The sensitivity was rescued by introducing functional SNM1, confirming its role.
Conclusions:
- Mammalian SNM1 plays a significant role in repairing DNA interstrand cross-links, particularly in response to mitomycin C.
- The function of SNM1 in ICL repair is conserved between yeast and mammals, although mammalian repair pathways exhibit redundancy.
- SNM1 deficiency in mice leads to sensitivity to ICL agents without causing major developmental abnormalities, highlighting its specific role in DNA repair.