Abstract B65: Protective role of RAD51D in resolving intermediates generated by mismatch repair recognition of DNA damage

Base mismatches can be generated during DNA replication or chemically induced by environmental genotoxic agents and anticancer chemotherapeutics. The anticancer agent 6‐thioguanine (6TG) and the known mutagen N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) cause me6TG and O 6 ‐meG lesions respectively, which mispair with thymine during DNA replication to cause G:C>A:T transitions. The DNA mismatch repair (MMR) pathway normally repairs mismatches caused by replication. However, recognition of me6TG:T/O 6 meG:T base mispairs by MutSα and MutLα complexes of MMR leads to a futile DNA repair cycle instead of repair, eventually resulting in apoptosis. Homologous recombination (HR) protects against killing induced by 6TG and MNNG treatments, but there is a gap in understanding the coordination among the MMR and HR pathways for resolving these lesions or eliminating damaged cells. To investigate MMR and HR genetic interactions, mice heterozygous for a null‐allele of the Mlh1 MMR gene and the Rad51d HR gene were crossed to generate offspring heterozygous at both loci. Phenotypes of embryos dissected from Rad51d +/− Mlh1 +/− intercrosses demonstrated that absence of Mlh1 did not alter the embryo lethal phenotype conferred by a Rad51d deletion. Absence of p53 ( Trp53 ) is required to permit development of Rad51d ‐deficient embryos to later stages and the isolation of Rad51d −/− cell lines. Therefore, mouse embryonic fibroblast (MEF) cell lines of Mlh1 −/− Rad51d −/− Trp53 −/− , Mlh1 −/− Trp53 −/− genotypes and each heterozygous combination were generated. Rad51d −/− Mlh1 +/+ MEFs were sensitive to MNNG and 6‐TG by 5‐fold compared to Rad51d +/+ Mlh1 +/+ MEFs. Mlh1 −/− MEFs were dramatically resistant to both MNNG and 6‐TG compared to Mlh1 +/+ MEFs. Interestingly, loss of MMR alleviated the sensitivity of Rad51d ‐deficient MEFs to 6‐TG and MNNG, which suggests MutSα/MutLα recognition of these mismatches causes a DNA intermediate resolved by HR. To further characterize the MMR‐HR genetic interaction, sensitivity of cells to the alkylating agent methyl methanesulfonate (MMS), which produces mostly N‐methyl purines and undetectable levels of O 6 ‐meG, was examined. Rad51d −/− MEFs were sensitized to MMS compared to Rad51d +/+ , regardless of Mlh1 status. These data further support the notion that MMR‐dependent sensitization of HR‐deficient cells is specific for me6TG/O 6 ‐meG lesions. In addition, MutSα/MutLα recognition of the base mispairs potently signaled G2/M cell cycle arrest and activated ATR/CHK1. Notably, the G2/M arrest was more pronounced in Rad51d −/− cells compared to Rad51d +/+ cells. This arrest was accompanied by an accumulation of double‐strand breaks and increased apoptosis, as measured by phosphorylation of H2AX and caspase‐3 activity, respectively. Also, a 10‐fold increase in chromosomal abnormalities was observed in the Rad51d −/− cells after 6‐TG treatment. Collectively, this work demonstrates that HR is required to protect against genome instability induced by MMR recognition of me6TG/O 6 ‐meG lesions and begins to elucidate the complex interplay between two DNA repair pathways following genotoxic insult. Citation Information: Cancer Res 2009;69(23 Suppl):B65.