It is the overall aim of this proposal to dissect the paradoxical mechanism by which the binding of a CAG hairpin converts an otherwise normal MMR complex into a mutational machine. Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. Surprisingly, the eukaryotic mismatch recognition complex, MSH2/MSH3, fails to act as a guardian of the genome and causes CAG expansion, the lethal mutation underlying Huntington's disease (HD) and more than 20 other neurodegenerative diseases. In this proposal, we focus on the two key mutagenic steps that cause the mutation: we will (1) determine why ATP hydrolysis in MSH2-MSH3 fails to signal loop removal, and (2) identify the endonuclease recruited by the MSH2-MSH3-hairpin complex that incorporates the loop into duplex DNA completing expansion.
In Aim 1 A, we will generate two ?separation-of-function? mutant KI mice for MSH2- MSH3, which bind ATP in each subunit, but lack ATP hydrolytic function in one or the other. If loss of hydrolytic activity in a particular subunit attenuates expansion, then the mutation requires the ATPase activity in that subunit.
In Aim 1 B, we will solve the crystal structure of MSH2-MSH3 bound to a repair competent (CA)4 loop or to the repair-resistant CAG hairpin. Identified are the structural perturbations in the nucleotide-bound MSH2- MSH3 complex that prevent proper removal of the hairpin loop.
In Aim 2, we will identify the canonical and non- canonical endonuclease machinery that facilitates incorporation of the hairpin loop and completes expansion. To identify non-canonical machinery, we will develop technology for site-specific capture of endonucleases ?caught in the act? of incising the loops at the CAG tract during expansion. Inserting a DNA site with CRISPR provides an engineered landing pad for targeting an engineered APEX2 fusion protein. The latter modifies closely located protein partners with biotin, which can be captured on streptavidin plates. We will test how these instructions are misinterpreted for ?in trans? nicking when MSH2-MSH3 is bound to the CAG hairpin. Collectively, the proposed experiments pave the way for small molecule development to restore loop removalby altering the hairpin DNA structure or the protein conformation.

Public Health Relevance

It is the overall aim of this proposal to dissect the paradoxical mechanism by which binding of a CAG hairpin converts an otherwise normal MMR complex into a mutational machine that caused more then 20 fatal trinucleotide expansion disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM119161-03
Application #
9766311
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Reddy, Michael K
Project Start
2017-08-01
Project End
2021-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720