RAD51D protects against MLH1-dependent cytotoxic responses to O6-methylguanine

Abstract

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 γ-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 γ-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HRdeficient 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.

Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

“…Even if the unreplicated regions go unrepaired and are converted to double strand breaks, the breaks could be a substrate for homologous recombination repair, again leading to cell survival. Consistent with this model, depletion of the homologous recombination repair protein RAD51D in mouse embryonic fibroblasts resulted in a 5-fold-increased sensitivity to MNNG compared with wildtype cells (45). This increased sensitivity was alleviated when MLH1 was also depleted, indicative of homologous recombination repair playing a role in resolving secondary damage generated by MMR lesion processing.…”

DNA mismatch repair and the DNA damage response

“…This has been observed in cells treated with alkylating drugs in which MGMT has been inhibited [71]. In another study, Rad51d Trp53-negative MEFs that are deficient for homologous recombination are hypersensitive to MNNG, but the additional deletion of Mlh1 resulting in loss of MMR partially rescues the sensitivity to MNNG [72]. This provides support for a role for homologous recombination in rescuing cells from otherwise lethal MMR-dependent processing of O 6 meG mismatches.…”

The Homologous Recombination Protein RAD51D Mediates the Processing of 6-Thioguanine Lesions Downstream of Mismatch Repair

“…Mouse embryonic fibroblast (MEF) cell lines Rad51d +/+ Trp53 -/- , Rad51d -/- Trp53 -/- , Rad51d -/- Trp53 -/- Mlh1 -/- and Mlh1 -/- Trp53 -/- were generated and characterized as described previously (12). Because Rad51d -/- MEFs with wild-type p53 fail to proliferate, all comparisons between MLH1 and RAD51D status were made with Trp53 -/- cells.…”

“…For example, 6-thioG induces sister chromatid exchanges (SCEs), a strong indicator of HR events, that are dependent on the presence of functional MMR (11). We recently reported the generation of a unique mouse model combining MMR and HR defects by deletion of Mlh1 and Rad51d (12). RAD51D is a member of the Rad51-protein family that plays an important role in HR, and has been identified to interact as part of a complex with MSH2 in a proteomics screen of RAD51D interacting partners (13).…”