Studies Of DNA Mismatch Repair

Environmental Health Sciences

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Abstract

Summary of Work: The goals of this project are to understand the biochemistry and genetics of MMR in normal eukaryotic cells, and how mutations in MMR genes lead to environmentally associated human diseases. This year we demonstrated that Mlh1-Pms1 binds and hydrolyzes ATP, and that its two ATPase active sites have different binding affinity and catalytic efficiency. We showed that the Mlh1-Pms1 heterodimer contains two separate DNA binding sites in the Mlh1 and Pms1 N-terminal domains and provided initial evidence suggesting that DNA binding by Mlh1 is important for MMR function. We showed that Mlh1 functions in germ cell development, and provide initial insights into the overlapping and/ separable roles of ATP binding, ATP hydrolysis and DNA binding on DNA transactions in mitotic versus meiotic cells. We showed that mouse Exo1 functions in mutation voidance and is essential for male and female meiosis. To test the hypothesis that the lower mutagenesis associated with replicating lagging strand templates is due to more efficient repair of lagging stand mismatches, we measured mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compared a MMR proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1 or EXOI. Loss of MMR reduced the strand bias by preferentially increasing mutagenesis for lagging strand replication, indicating that 8-O-G?A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, both present at higher density during lagging strand replication, are used as strand discrimination signals, thus providing the first evidence for the identity of this signal in vivo in a eukaryotic cell. In collaboration with the Resnick group, we provide evidence that cadmium inhibits MMR activity in yeast and in human cell free extracts.