Publication Date

5-2017

Advisor(s)

Manju M. Hingorani

Department

Molecular Biology & Biochemistry

Language

English

Abstract

The mismatch repair (MMR) pathway is responsible for correcting errors in DNA and is essential for the maintenance of genome stability in all living organisms. Several studies have shown that mutations adversely affecting the structure and function of MMR proteins result in a higher frequency of mutations within the genome. Such mutations have been linked to disease phenotypes such as Lynch Syndrome, a hereditary predisposition to colorectal and other cancers. The first protein in the MMR pathway, MutS, recognizes a mismatch or insertion-deletion loop and initiates DNA repair using its ATPase activity. MutS mutations located in the DNA binding and ATPase sites can be expected to disrupt these critical activities; however, there are also several reported mutations located far from the active sites and it is not immediately clear how these mutations beget the disease phenotype. This study utilizes biochemical techniques to determine whether and how these mutations change the MutS mechanism and in turn, disrupt MMR. Preliminary kinetic analysis was carried out on five T. aquaticus MutS mutants (L166R, F243S, I400G, G434R, L602R) that are homologous to human MutSa mutants identified in Lynch Syndrome patients. For each mutant, fluorescence reporter assays were used to monitor the transient kinetics of interaction with a +T insertion in DNA and the ATPase activity under pre-steady state conditions. The results identified key steps in the MutS mechanism affected by the cancer-linked mutations: G434R and L602R associate with the +T insertion faster than WT MutS, resulting in an increased affinity for the error; I400G and L602R dissociate differently from the +T insertion in the presence of ATP, indicating that they undergo different ATPinduced conformational changes compared to WT; L602R disrupts ATP hydrolysis; and L166R and I400G ATPase activity appears partially responsive or unresponsive to the +T insertion, indicating uncoupling of DNA binding and ATPase activities. These differences in the MutS mechanism potentially result in defective MMR. Overall, the study demonstrates how a detailed biochemical analysis can provide a basis for understanding the mechanisms of Lynch Syndrome at the molecular level.

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