Date

May 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular and Medical Genetics

Institution

Oregon Health & Science University

Abstract

DNA mismatch repair (MMR) is a versatile cellular mechanism that corrects errors formed during replication, responds to certain types of DNA damage, and helps control recombination. When mismatches arise in DNA due to polymerase errors, MMR responds by assembling a multi-protein complex that detects and removes the mismatch. This complex is also necessary to signal apoptosis in response to certain types of DNA damage. Central to MMR’s function in mutation avoidance is the heterodimer MutLα, composed of MLH1 and PMS2, which acts to couple mismatch recognition to downstream steps. Both MLH1 and PMS2 contain ATPase domains in their N-termini, and it had previously been shown in yeast and in human in vitro analyses that MutLα ATP binding and hydrolysis is necessary for repair. However, the relative contributions of each ATPase domain to repair had yet to be examined in vivo in mammalian cells. I analyzed the effect of mutations in the highly conserved ATPase domain of MutLα in vivo in mouse cell culture. I observed that mutations impacting ATP binding and hydrolysis in the MutLα protein MLH1 impact repair to a greater degree than the equivalent ATPase domain mutations in MLH1’s binding partner PMS2, as measured by instability at microsatellite loci. I also examined the effect of the mutations on the cytotoxic response to the methylation mimetic 6-thioguanine (6-TG), which elicits a MMR-dependent apoptotic response. Consistent with the mutator results, mutations in the ATPase domain of MLH1 caused resistance to 6-TG, while mutations in the ATPase domain of PMS2 did not. These results indicate a functional asymmetry in the contributions of the ATPase domains of the MutLα partners to repair and that MLH1 and PMS2 may have distinct roles during repair. In the second part of my research, I examined the role of the nucleotide pool regulator deoxycytidylate deaminase (DCTD) in the MMR-dependent response to 6-TG. Previous work with the yeast homologue DCD1 had shown that DCD1 plays a role in this response. DCTD catalyzes the conversion of dCMP to dUMP and thus helps maintain the dCTP:dTTP balance within the cell. I found that reduced expression of DCTD in human cells causes increased resistance to 6-TG, indicating DCTD is a necessary component in the MMR-dependent response to 6-TG in human cells. Surprisingly, cells derived from Dctdko/ko mice do not display significant resistance to 6-TG. When nucleotide pool levels were measured, the cells derived from the Dctdko/ko mice did not exhibit as significant dCTP:dTTP imbalance as previously reported in established rodent cell lines. In addition, the human cells displayed no significant imbalance. These results suggest an adaptive response to maintaining the dCTP:dTTP pool levels in the absence of Dctd in the mouse, and also suggest that resistance to 6-TG toxicity in human cells can occur without dCTP:dTTP pool imbalance.

Identifier

doi:10.6083/M4K935H4

School

School of Medicine

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