There is a fundamental gap in our understanding of how mutations in enzymes of the base excision repair (BER) pathway may affect a protein's function, global conformation and its interactions with protein partners, and how these changes can lead to initiation of carcinogenesis. A powerful combination of structural, biochemical and cellular methods will be employed to study the molecular mechanisms of the human BER glycosylases that repair oxidative damage. These enzymes are the ?first responders? as their task is to recognize and excise oxidized bases in DNA while leaving normal bases untouched. The central hypothesis of this program project is that defects in BER proteins can drive human carcinogenesis and affect responses to cancer treatments. The objective of Project 2 is to understand, at the biochemical and structural levels, how the BER glycosylases recognize and process oxidized lesions, how the flexible regions of the proteins influence activity and interactions with DNA or protein partners, and how single-point mutations affect the protein form and function and may ultimately initiate carcinogenesis. Guided by strong preliminary data the three aims of this proposal will 1- determine the biochemical and molecular mechanisms of lesion recognition by the NEIL glycosylases, 2- elucidate the molecular mechanisms of inhibition, activation and dimerization of NTHL1 glycosylase, and 3- evaluate the effects of BER glycosylase mutations by determining the biochemical and structural characteristics of these variants and assessing their biological phenotypes.
These aims will use biochemical and structural biology methods, such as X-ray crystallography and small angle X-ray scattering (SAXS), which will be used to determine the shape and form of the full-length glycosylases and potential changes brought upon by mutations. The structure/function studies from Project 2 will work synergistically with the phenotypical characterization in human cells carried out by Project 1. Our work also dovetails with the work done in Project 3 on NTHL1 and substrate hand off, and the single-molecule studies carried out by Project 4. Core A will provide bioinformatics and statistical support for the study of the human variants. Purified proteins and human cell cultures will be provided by Core B. We anticipate that this work will provide fundamental insights into the molecular mechanisms of BER glycosylases. These results are expected to have a positive impact because they will reveal how amino acid substitutions in DNA glycosylases lead to initiation of carcinogenesis, knowledge that will be beneficial for predicting cancer susceptibility and optimizing treatment strategies.

Public Health Relevance

These biochemical and structural biology studies will advance our understanding of how DNA repair enzymes recognize and repair DNA lesions and how mutations in these enzymes contribute to individual cancer risk. Understanding how mutations in DNA glycosylases affect their function will inform prognosis and cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
2P01CA098993-11A1
Application #
9209396
Study Section
Special Emphasis Panel (ZCA1-RPRB-F (O1))
Project Start
2004-09-03
Project End
2022-04-30
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
11
Fiscal Year
2017
Total Cost
$345,300
Indirect Cost
$121,645
Name
University of Vermont & St Agric College
Department
Type
Domestic Higher Education
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Galick, Heather A; Marsden, Carolyn G; Kathe, Scott et al. (2017) The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation. Oncotarget 8:85883-85895
Silva, Michelle C; Bryan, Katie E; Morrical, Milagros D et al. (2017) Defects in recombination activity caused by somatic and germline mutations in the multimerization/BRCA2 binding region of human RAD51 protein. DNA Repair (Amst) 60:64-76
Robey-Bond, Susan M; Benson, Meredith A; Barrantes-Reynolds, Ramiro et al. (2017) Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues. DNA Repair (Amst) 53:43-51
Maher, R L; Marsden, C G; Averill, A M et al. (2017) Human cells contain a factor that facilitates the DNA glycosylase-mediated excision of oxidized bases from occluded sites in nucleosomes. DNA Repair (Amst) 57:91-97
Cannan, Wendy J; Rashid, Ishtiaque; Tomkinson, Alan E et al. (2017) The Human Ligase III?-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA. J Biol Chem 292:5227-5238
Zhou, Jia; Chan, Jany; Lambelé, Marie et al. (2017) NEIL3 Repairs Telomere Damage during S Phase to Secure Chromosome Segregation at Mitosis. Cell Rep 20:2044-2056
Marsden, Carolyn G; Jensen, Ryan B; Zagelbaum, Jennifer et al. (2016) The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype. PLoS Genet 12:e1006208
Lee, Andrea J; Wallace, Susan S (2016) Visualizing the Search for Radiation-damaged DNA Bases in Real Time. Radiat Phys Chem Oxf Engl 1993 128:126-133
Cannan, Wendy J; Pederson, David S (2016) Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin. J Cell Physiol 231:3-14
Silva, Michelle C; Morrical, Milagros D; Bryan, Katie E et al. (2016) RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects. DNA Repair (Amst) 42:44-55

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