Abstract B65: Protective role of RAD51D in resolving intermediates generated by mismatch repair recognition of DNA damage
Authors: Rajesh Preeti , Litvinchuk Alexandra , Rajesh Changanamkandath , Pittman Douglas L. , Wyatt Michael D. 
Published: May 2012

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 O6-meG lesions respectively, which mispair with thymine during DNA replication to cause G:C&gt;A:T transitions. The DNA mismatch repair (MMR) pathway normally repairs mismatches caused by replication. However, recognition of me6TG:T/O6meG: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 O6-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/O6-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/O6-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.