In order to provide insight into how the oxidative DNA glycosylases may malfunction and thus lead to genome instability and cause cancer, we propose to use structural, biochemical and biological approaches to elucidate how these enzymes perform the first step In base excision repair. The long-term goal of this project is to not only provide fundamental insights into the mechanisms underpinning carcinogenesis but also to inform the human germ line SNP databases and allow us to predict with greater accuracy which SNP may lead to an increased risk of cancer. Moreover, understanding how variants In human tumors may affect function will inform prognosis and cancer treatment. The specific alms are as follows:
Aim 1, A- To solve the crystal structures of the human members of the Fpg/Nei family NEIL1, NEIL2, and NEIL3, or close orthologs such as the mimivirus Nei proteins in complex with their DNA substrates. Towards this goal we have already obtained complexes of Mimivirus Neil with thymine glycol and spiroiminodihydantoin, which are the first examples of any Nei enzyme in complex with an oxidative DNA lesion. B- Once the structures are determined with substrate in the binding pocket, site directed mutants will be constructed for further biochemical analyses. C- To test the hypothesis that the NEILI, 2 and 3 variants Identified In Project 1 and Core A contribute to carcinogenesis by determining the biochemical characteristics of these variants such as substrate specificity and DNA binding properties and, where Indicated, their crystal structures.
Aim 2. A- To solve the crystal structures of human members of the Nth superfamily or close orthologs in complex with their DNA substrates. Of note Is that no one has yet been able to crystallize hNTHI. B- To examine a select group of hNTHI germ line variants identified in Project 1 and Core A and through biochemical and structural analysis determine which variants may have potentially deleterious consequences. Structure/function studies from Project 2 will Inform the biological studies in Project 1 and provide insight into the interactions of glycosylases with nucleosomes In Project 4, Purified proteins and rapid enzyme analysis will be provided by Core B. Core A will help design and analyze enzyme kinetics experiments,

Public Health Relevance

These studies will advance our understanding of how DNA repair variants contribute to individual cancer risk and how they drive carcinogenesis. Moreover, the oxidative DNA glycosylases may be exploited as targets for increasing therapeutic efficacy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA098993-09
Application #
8543549
Study Section
Special Emphasis Panel (ZCA1-RPRB-0)
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
9
Fiscal Year
2013
Total Cost
$363,410
Indirect Cost
$89,516
Name
University of Vermont & St Agric College
Department
Type
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Prakash, Aishwarya; Cao, Vy Bao; Doublié, Sylvie (2016) Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase. PLoS One 11:e0157860
Cannan, Wendy J; Pederson, David S (2016) Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin. J Cell Physiol 231:3-14
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
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
Zhou, Jia; Fleming, Aaron M; Averill, April M et al. (2015) The NEIL glycosylases remove oxidized guanine lesions from telomeric and promoter quadruplex DNA structures. Nucleic Acids Res 43:4039-54
Chen, Jianhong; Morrical, Milagros D; Donigan, Katherine A et al. (2015) Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase. Nucleic Acids Res 43:1098-111
Morrical, Scott W (2015) DNA-pairing and annealing processes in homologous recombination and homology-directed repair. Cold Spring Harb Perspect Biol 7:a016444
Prakash, Aishwarya; Doublié, Sylvie (2015) Base Excision Repair in the Mitochondria. J Cell Biochem 116:1490-9
Prakash, Aishwarya; Carroll, Brittany L; Sweasy, Joann B et al. (2014) Genome and cancer single nucleotide polymorphisms of the human NEIL1 DNA glycosylase: activity, structure, and the effect of editing. DNA Repair (Amst) 14:17-26

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