DNA mismatch repair is a major contributor to genetic stability. Mismatch repair defects confer strong cancer predisposition and have implications for cancer therapy because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs, a consequence of participation of the system in the DNA damage response. Perhaps surprisingly, mismatch repair function is also required for production of certain mutations, such as the expansion of (CAG){n} repeat sequences, the primary cause of a number of neurodegenerative diseases. The goals of this project to clarify conformations, conformational variation, and structures of multi-protein, protein-DNA, and multi-protein DNA assemblies that are key intermediates in DNA lesion processing and damage signaling by the mismatch repair system.
Our aims are 3-fold: (1) Using deuterium exchange mass spectrometry (DXMS), we have identified regions of bacterial MutS and eukaryotic MutS-alpha (MSH2-MSH6) that undergo substrate-dependent conformational transitions. These regions will be subjected to site-directed mutagenesis and the resulting mutants characterized for their impact on mismatch repair both in vivo and in vitro. (2) Small angle X-ray scattering, equilibrium methods, DXMS, x-ray crystallographic, biochemical and genetic approaches as appropriate will be utilized to extend our understanding of multi-protein assemblies involved in mismatch repair. These assemblies will include PCNA complexes with MutS-alpha (MSH2-MSH6) and MutL-alpha (MLH1-PMS1/PMS2), MutSa complexes with exonuclease 1 and with Chk1, and the complex between exonuclease 1 and the BLM helicase. The latter study will be pursued in collaboration with Project 4. (3) Human MutS-alpha and MutS-Beta differ in the manner in which they interact with PCNA and MutL-alpha, indicating that MutS-alpha- and MutS-Beta-triggered repair events proceed by significantly different mechanisms. In view of the known involvement of MutS-Beta and MutL-alpha in the somatic phase (CAG){n}:(CTG){n}, triplet repeat expansion, we will seek small molecule inhibitors that specifically block MutS Beta-triggered repair events by screening for compounds that selectively block assembly the MutL-alpha-MutS-Beta-DNA ternary complex.

Public Health Relevance

DNA mismatch repair provides multiple mutation avoidance functions. Inactivation of the pathway is the cause of both inherited and sporadic cancers, but also renders tumor cells resistant to certain chemotherapeutic regimens. The goals of this project are to clarify conformations and structures of multi-protein, protein-DNA, and multi-protein-DNA assemblies that are intermediates in lesion processing by this DNA repair system, providing insights into a pathway that impacts the development and treatment of cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA092584-14
Application #
8728565
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
14
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
City
Berkeley
State
CA
Country
United States
Zip Code
Sengupta, Shiladitya; Yang, Chunying; Hegde, Muralidhar L et al. (2018) Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress. DNA Repair (Amst) 66-67:1-10
Mu, Hong; Geacintov, Nicholas E; Broyde, Suse et al. (2018) Molecular basis for damage recognition and verification by XPC-RAD23B and TFIIH in nucleotide excision repair. DNA Repair (Amst) :
Chavez, Diana A; Greer, Briana H; Eichman, Brandt F (2018) The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression. J Biol Chem 293:8484-8494
Wang, Jing L; Duboc, Camille; Wu, Qian et al. (2018) Dissection of DNA double-strand-break repair using novel single-molecule forceps. Nat Struct Mol Biol 25:482-487
Crickard, J Brooks; Kaniecki, Kyle; Kwon, Youngho et al. (2018) Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase. Proc Natl Acad Sci U S A 115:E10041-E10048
Syed, Aleem; Tainer, John A (2018) The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair. Annu Rev Biochem 87:263-294
Howes, Timothy R L; Sallmyr, Annahita; Brooks, Rhys et al. (2018) Erratum to ""Structure-activity relationships among DNA ligase inhibitors; characterization of a selective uncompetitive DNA ligase I inhibitor"" [DNA Repair 60C (2017) 29-39]. DNA Repair (Amst) 61:99
Bhattacharyya, Sudipta; Soniat, Michael M; Walker, David et al. (2018) Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase. Proc Natl Acad Sci U S A 115:E11614-E11622
Tsai, Chi-Lin; Tainer, John A (2018) Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes. Methods Enzymol 599:157-196
Ogorzalek, Tadeusz L; Hura, Greg L; Belsom, Adam et al. (2018) Small angle X-ray scattering and cross-linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy. Proteins 86 Suppl 1:202-214

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