Publication Date

5-2017

Advisor(s)

Manju M. Hingorani

Department

Molecular Biology and Biochemistry

Language

English

Abstract

Mismatch repair (MMR) is a crucial process that corrects errors produced by DNA polymerase. During DNA replication, the polymerase can incorporate an incorrect base or slip on the primer-template junction, leading to base-pair mismatches and insertion-deletion loops (IDLs), respectively. In eukaryotes, MutS homologs Msh2-Msh6 and Msh2-Msh3 recognize these errors; Msh2-Msh6 binds base-pair mismatches and smaller IDLs, and Msh2-Msh3 binds larger IDLs. Following error recognition, downstream proteins are recruited to repair the errorcontaining DNA, thus promoting genomic stability. Notably, the actions of Msh2- Msh3 on DNA can also lead to genomic instability. During replication, the polymerase is likely to slip on triplet nucleotide repeats (TNRs) and form hairpin structures. Msh2-Msh3 is thought to stabilize these hairpins and promote TNR expansion, which has been associated with various neurological disorders such as Huntington’s disease. Surprisingly, Msh2-Msh6 does not appear to promote TNR expansion in most disease models and may protect against TNR contractions. Studying the mechanistic differences between Msh2-Msh3 and Msh2-Msh6 actions on TNR hairpin structures will help with understanding their differential effects on TNR instability. This project aims to understand how Msh2-Msh6 recognizes and responds to its cognate error-containing DNA substrates compared to TNR hairpins. Gel mobility shift assays confirmed that Msh2-Msh6 forms a tight complex with a CAG hairpin, similar to base-pair mismatches, leading to the hypothesis that Msh2- Msh6 recognizes the A:A mismatches within the TNR hairpin stem. To test this hypothesis, Msh2-Msh6 ATPase activity was compared with error-containing DNA and TNR hairpin structures. Pre-steady state ATPase measurements indicate that Msh2-Msh6 responds to a CAG hairpin structure like a mismatch. Thus, although Msh2-Msh6 binds CAG hairpins with high affinity, it may slide off in an abortive attempt to initiate repair and therefore does not stabilize this DNA structure like Msh2-Msh3. Further kinetic analysis of Msh2-Msh6 interactions with TNR hairpins, including the effects of nucleotides, and its subsequent interaction with Mlh1-Pms1 is necessary to test this prediction. Together with analogous studies of Msh2-Msh3 in the future, these results may help resolve why Msh2-Msh6 does not promote TNR expansion and suggest ways to prevent the development of TNR-based neurological disorders.

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