RAD51D protects against MLH1-dependent cytotoxic responses to O(6)-methylguanine.
S(N)1-type methylating agents generate O(6)-methyl guanine (O(6)-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O(6)-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR-dependent mutagenicity and toxicity caused by O(6)-meG has been studied, the mechanisms of recombination induced by O(6)-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d(-/-)Mlh1(-/-)Trp53(-/-) MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3-fold sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of gamma-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing gamma-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O(6)-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O(6)-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O(6)-meG adducts.
Department of Pharmaceutical and Biomedical Sciences, University of South Carolina, Columbia, 29208, United States.
Preeti
P
Rajesh
Changanamkandath
C
Rajesh
Michael D
MD
Wyatt
Douglas L
DL
Pittman
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004622:Embryo, Mammalian / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D016195:G2 Phase; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D051379:Mice; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000235:genetics / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C501485:Mlh1 protein, mouse; D009687:Nuclear Proteins; C491807:Rad51d protein, mouse; D008769:Methylnitronitrosoguanidine; D006147:Guanine; C008449:O-(6)-methylguanine; D051135:Rad51 Recombinase; D000070957:MutL Protein Homolog 1