Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. We have surprisingly found that the eukaryotic mismatch recognition complex, MSH2/MSH3, not only fails to act as a guardian of the genome, but also causes CAG expansion, the lethal mutation underlying Huntington's disease (HD). It is the overall aim of this renewal to build on these discoveries, and to dissect the paradoxical mechanism by which binding of a CAG hairpin converts functional MSH2/MSH3 into a defective machine that causes mutation and disease. Although the molecular details of MutSa function are still incompletely understood, it is clear that some form of conformational coupling between DNA recognition and the nucleotide binding sites plays a central role. Our preliminary data indicate that MSH2/MSH3 binds with high affinity to the CAG hairpin, but binding there inhibits its ATPase activity, alters nucleotide affinity, and prevents translocation along DNA relative to repair competent loops. In order to define how the disease-causing CAG-hairpin diverts the MSH2MSH3 protein from normal repair, it becomes imperative to define the DNA and nucleotide dependent properties of the repair-competent MSH2MSH3-DNA complexes. The biochemical characterization of MSH2/MSH3 has lagged far behind that of MSH2/MSH6, and many parameters are unknown. MSH2/MSH3 is different in the way it recognizes DNA, and has different lesion specificity, and the sites of nucleotide binding in MSH2 and MSH3 subunits have not yet been mapped. Despite key differences, the biochemical properties of MSH2/MSH3 have been primarily extrapolated from MutS and its mammalian homologue, MSH2/MSH6. We propose two specific aims that elucidate the mechanism of uncoupling and how dissociation of DNA binding and ATP hydrolysis in MSH2/MSH3 leads to CAG expansion and disease.
In Aim 1, we create a dynamic system for visualizing DNA and nucleotide dynamics in MSH2/MSh3-CAG hairpin complexes. Using combined smFRET and a panel of biochemical methods, We establish a real time system to monitor the relationships between DNA bindings, nucleotide binding and translocation of MSH2/MSh3 on repair competent and repair deficient substrates.
In Aim2, we use SAX analysis and limited proteolysis to probe the DNA-induced conformational changes imposed on MSH2/MSH3 by hairpins binding. We will test whether those conformational alterations determine template specificity and influence in protein interactions that divert MSH2/MSh3 function in MMR. These two aims integrate protein biochemistry, nucleotide dynamics, and conformational analysis with in vivo biology in primary animals cells reflecting the disease. These studies will not only provide insights into how mutations in the mismatch repair genes cause expansion and disease, but will also broaden our conceptual framework for DNA damage recognition.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM066359-06
Application #
7596301
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Krasnewich, Donna M
Project Start
2004-04-01
Project End
2012-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
6
Fiscal Year
2009
Total Cost
$429,916
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Genetics
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Lee, Do Yup; Xun, Zhiyin; Platt, Virginia et al. (2013) Distinct pools of non-glycolytic substrates differentiate brain regions and prime region-specific responses of mitochondria. PLoS One 8:e68831
Hura, Greg L; Budworth, Helen; Dyer, Kevin N et al. (2013) Comprehensive macromolecular conformations mapped by quantitative SAXS analyses. Nat Methods 10:453-4
Budworth, Helen; McMurray, Cynthia T (2013) Bidirectional transcription of trinucleotide repeats: roles for excision repair. DNA Repair (Amst) 12:672-84
Budworth, Helen; McMurray, Cynthia T (2013) A brief history of triplet repeat diseases. Methods Mol Biol 1010:3-17
Xun, Zhiyin; Rivera-Sánchez, Sulay; Ayala-Peña, Sylvette et al. (2012) Targeting of XJB-5-131 to mitochondria suppresses oxidative DNA damage and motor decline in a mouse model of Huntington's disease. Cell Rep 2:1137-42
Majka, Jerzy; Alford, Brian; Ausio, Juan et al. (2012) ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease. J Biol Chem 287:2328-41
Lang, Walter H; Coats, Julie E; Majka, Jerzy et al. (2011) Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops. Proc Natl Acad Sci U S A 108:E837-44
Kovtun, Irina V; Johnson, Kurt O; McMurray, Cynthia T (2011) Cockayne syndrome B protein antagonizes OGG1 in modulating CAG repeat length in vivo. Aging (Albany NY) 3:509-14
McMurray, Cynthia T (2010) Mechanisms of trinucleotide repeat instability during human development. Nat Rev Genet 11:786-99
Owen, Barbara A L; H Lang, Walter; McMurray, Cynthia T (2009) The nucleotide binding dynamics of human MSH2-MSH3 are lesion dependent. Nat Struct Mol Biol 16:550-7

Showing the most recent 10 out of 22 publications