Year of Publication

2010

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Medicine

Department

Toxicology

First Advisor

Dr. Guo-min Li

Abstract

The integrity of an organism's genome depends on the fidelity of DNA replication and the efficiency of DNA repair. The DNA mismatch repair (MMR) system, which is highly conserved from prokaryotes to eukaryotes, plays an important role in maintaining genome stability by correcting base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication and other DNA transactions. Mismatch recognition is a critical step in MMR. Two mismatch recognition proteins, MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer), have been identified in eukaryotic cells. MutSα and MutSβ have partially overlapping functions, with MutSα recognizing primarily base-base mismatches and 1-2 nt ID mispairs and MutSβ recognizing 2-16-nt ID heteroduplexes. The goal of this dissertation research was to understand the mechanism underlying differential mismatch recognition by human MutSα and MutSβ and to characterize the unique functions of human MutSα and MutSβ in MMR.

In this study, recombinant human MutSα and MutSβ were purified. Binding of the proteins to a T-G mispair and a 2-nt ID mispair was analyzed by gel-mobility assay; ATP/ADP binding was characterized using a UV cross-linking assay; ATPase activity was measured using an ATPase assay; MutSα amd MutSβ’s mismatch repair activity was evaluated using a reconstituted in vitro MMR assay.

Our studies revealed that the preferential processing of base-base and ID heteroduplexes by MutSα and MutSβ respectively, is determined by the significant differences in the ATPase and ADP binding activities of MutSα and MutSβ, and the high ratio of MutSα:MutSβ in human cells. Our studies also demonstrated that MutSβ interacts similarly with a (CAG)n hairpin and a mismatch, and that excess MutSβ does not inhibit (CAG)n hairpin repair in vitro.

These studies provide insight into the determinants of the differential DNA repair specificity of MutSα and MutSβ, the mechanism of mismatch repair initiation, and the mechanism of (CAG)n hairpin processing and repair, which plays a role in the etiology and progression of several human neurological diseases.

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