msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

Kijas, Amanda Wraith, Studamire, Barbara and Alani, Eric (2003) msh2 separation of function mutations confer defects in the initiation steps of mismatch repair. Journal of Molecular Biology, 331 1: 123-138. doi:10.1016/S0022-2836(03)00694-6


Author Kijas, Amanda Wraith
Studamire, Barbara
Alani, Eric
Title msh2 separation of function mutations confer defects in the initiation steps of mismatch repair
Formatted title
msh2 separation of function mutations confer defects in the initiation steps of mismatch repair
Journal name Journal of Molecular Biology   Check publisher's open access policy
ISSN 0022-2836
1089-8638
Publication date 2003-08-01
Sub-type Article (original research)
DOI 10.1016/S0022-2836(03)00694-6
Open Access Status
Volume 331
Issue 1
Start page 123
End page 138
Total pages 16
Place of publication London, United Kingdom
Publisher Academic Press
Language eng
Formatted abstract
In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3′ non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.
Keyword MSH2-MSH6
MLH1-PMS1
Mismatch repair
ATPase
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: UQ Centre for Clinical Research Publications
 
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Created: Tue, 17 Jun 2014, 11:46:42 EST by Amanda W. Kijas on behalf of UQ Centre for Clinical Research