Exo1 Independent DNA Mismatch Repair Pathway in Hematopoiesis.

Abstract 2526

Poster Board II-503

The DNA mismatch repair pathway (MMR) is a fundamental process in cells that functions to correct mispaired bases and insertion/deletion loops caused by errors in replication. Failure in MMR can lead to the accumulation of mutations and carcinogenesis, notably hereditary nonpolyposis colorectal cancer (HNPCC). In hematopoiesis, loss of MMR results in methylating agent resistance and an HSC repopulation defect. Our research focused on the significance of Exonuclease 1, the enzyme responsible for excising mispaired bases in MMR. Interestingly Exo1^−/− mice display a much milder pathogenic phenotype when compared to the MMR deficient MSH2^−/− mice; characterized by lower mutation rates, lower levels of microsatellite instability, and in humans no association with HNPCC. These findings led us to hypothesize that the limited phenotype observed in Exo1−/− mice is due to (a) complementation through another exonuclease occurs which restores the proficiency of the MMR pathway or (b) an alternative pathway exists which allows for exonuclease independent repair.

To test this hypothesis we derived primary MEFs from WT, Exo1^−/− and MSH2^−/− mice and performed temozolomide sensitivity assays. We observed that Exo1^−/− MEFs are similarly sensitive as WT cells when treated with the drug, confirming proficient MMR, while MSH2^−/− MEFs display a decreased sensitivity. Additionally, comet assays with the same cell populations show a persistence of DNA single strand breaks in the Exo1^−/− and WT cells 24 hours post temozolomide treatment, consistent with proficient MMR activity, while MSH2^−/− MEFs show no such damage. To confirm functional MMR in hematopoiesis of Exo1^−/− mice we conducted a competitive repopulation study. We found that Exo1^−/− and WT marrow engraft at similar levels 8 and 16 weeks post transplantation. We subsequently treated these mice with 80 mg/kg temozolomide to determine if Exo1^−/− marrow progenitors would confer a competitive survival advantage post treatment. Consistent with our hypothesis we found that the ratio of marrow cells remained approximately 50:50 (Exo1:WT) in contrast to data obtained from MSH2^−/− mice in which the MSH2^−/− cells display a strong 95:5 (MSH2:WT) advantage post drug treatment. This strongly suggests functional MMR in the Exo1 deficient mice. We next coimmunoprecipitated the MMR complex by pulling down Mlh1 in Exo1^−/− and WT MEFs. Exo1, a key exonuclease in mismatch repair, does not modulate hematopoietic function as do the other MMR proteins and appears to be complemented by an as yet undefined exonuclease.