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. 2010 Apr 4;9(4):458-67.
doi: 10.1016/j.dnarep.2010.01.009. Epub 2010 Feb 4.

RAD51D protects against MLH1-dependent cytotoxic responses to O(6)-methylguanine

Preeti Rajesh  1 , Changanamkandath Rajesh, Michael D Wyatt, Douglas L Pittman
Affiliations

RAD51D protects against MLH1-dependent cytotoxic responses to O(6)-methylguanine

Preeti Rajesh et al. DNA Repair (Amst). .

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 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.

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Figures

Fig. 1
Fig. 1
An Mlh1 deletion does not affect the phenotype of Rad51d null embryos. (A & B) Representative E11.5 (A) and E15.5 (B) embryos in an order of wild-type, Rad51d−/−Mlh1−/− and Rad51d−/−Mlh1−/− in Trp53+/+ and Trp53−/− background respectively. (C) Genotypes of the embryos as determined by PCR. For the Rad51d gene, 285- and 250-bp fragments identify the disrupted and wild-type alleles, respectively. For the Trp53 gene, 600- and 450-bp fragments identify the disrupted and wild-type alleles, respectively. For the Mlh1 gene, 198- and 258-bp fragments identify the disrupted and wild-type alleles, respectively. (D) MGMT protein expression was analyzed in Mlh1−/−Trp53−/−, Rad51d−/−Trp53−/−Mlh1−/−, Rad51d−/−Trp53−/−, Trp53−/−, and wild-type MEFs by Western blot. β-Actin was used as a loading control.
Fig. 2
Fig. 2
RAD51D is required for resistance to O6-meG lesions in MMR-proficient MEFs. Colony survival assays of MEFs deficient in Rad51d, Mlh1 or both, treated with MNNG at the indicated doses. (A) Pair-wise comparison of Rad51d-proficient (♦) and Rad51d-deficient (■) MEFs. (B) Pair-wise comparison of Mlh1-proficient (♦) and Mlh1-deficient MEF (○). (C) Pair-wise comparison of Rad51d-deficient Mlh1-proficient (■) and Rad51d-deficient Mlh1-deficient (▲) MEFs. Clonogenic survival assays show that deleting Mlh1 in Rad51d-deficient cells rescues the sensitivity of these cells to MNNG 5.2-fold. Survival is plotted as percentage compared to untreated control. Each data point represents the mean of three independent experiments each performed in triplicates. Error bars represent the standard error of means. All MEFs are Trp53−/−.
Fig. 3
Fig. 3
Sensitivity of Rad51d-deficient MEFs to MMS is independent of MMR status. Colony survival assays of MEFs deficient in Rad51d, Mlh1 or both, treated with MMS at the indicated doses. MLH1 status does not affect the sensitivity of Rad51d-proficient or Rad51d-deficient MEFs in response to MMS [Rad51d and Mlh1-proficient (♦), Rad51d-proficient and Mlh1-deficient (○), Rad51d-deficient and Mlh1-proficient (■) and Rad51d-deficient and Mlh1-deficient (▲)]. Survival is plotted as percentage compared to untreated control. Each data point represents the mean of three independent experiments each performed in triplicates. Error bars represent the standard error of means. All MEFs are Trp53−/−.
Fig. 4
Fig. 4
Cell cycle arrest of Rad51d-deficient MEFs in response to MNNG is Mlh1 dependent. Percentage of the cells in G2/M phase is shown for (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/− and (D) Rad51d−/−Mlh1−/− MEFs at indicated time points after MNNG treatment. Progression of cell cycle was followed for untreated cells and those treated with 1 µM MNNG (■ Control and □ Treatment). Percentage of the cells in the G2/M phase was calculated using MODFIT-LT software. Each data point represents the mean of three independent experiments. Error bars represent the standard error of means. Statistical significance was determined by ratio t-test (*P<0.05). All MEFs are Trp53−/−.
Fig. 5
Fig. 5
Activation of Caspase-3 in Rad51d and Mlh1 deficient MEFs in response to MNNG treatment. (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/−, and (D) Rad51d −/−Mlh1−/− MEFs were treated with 1 µM MNNG for the indicated times. After treatment, cells were lysed and assayed for caspase-3 activity using EnzChek Caspase-3 Assay kit (Invitrogen) (■ Control and □ Treatment). The experiment was performed in triplicates and the data represents mean±s.d. Statistical significance was determined by Student’s t-test (*P<0.05). All MEFs are Trp53−/−.
Fig. 6
Fig. 6
Kinetics of H2AX phosphorylation in MNNG treated MEFs. (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/-Mlh1+/+, (C) Rad51d+/+Mlh1−/− and (D) Rad51d−/−Mlh1−/− MEFs were treated with 1 µM MNNG for the indicated times. Total cell lysates were analyzed for phosphorylated H2AX (γ-H2AX) by western blot analysis. β-Actin was used as a control for equal protein loading. The bands visualized via western blot were subjected to band densitometry analysis using Image J software (open source Image J software available at http://rsb.info.nih.gov/ij/). The amount of specific signal for γ-H2AX was corrected for sample loading by normalization with the constitutive β-Actin signal. The value for the 0 h time point was set as 1 to calculate the Fold Induction values which were plotted as a function of time.
Fig. 7
Fig. 7
Kinetics of CHK1 phosphorylation in Rad51d and Mlh1 deficient MEFs in response to treatment with 1µM MNNG. (A) Rad51d+/+Mlh1+/+, (B) Rad51d−/−Mlh1+/+, (C) Rad51d+/+Mlh1−/− and (D) Rad51d−/−Mlh1−/− MEFs were treated with 1 µM MNNG for the indicated times. Whole cell lysates were analyzed for total CHK1, phospho-CHK1(Ser345), by western blot analysis. β-Actin was used as a control for equal protein loading. The bands visualized via western blot were quantified by Image J software (open source Image J software available at http://rsb.info.nih.gov/ij/). The amount of p-CHK1 relative to total CHK1 was calculated as ratio.
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