Every day, normal cellular metabolism produces 30,000 or more oxidative lesions In the DNA of each human cell. Most of these lesions are removed by the base excision repair (BER) machinery. Certain defective BER enzymes have been shown to be oncogenic, Nucleosomes that package DNA into chromafin provide little if any protection from oxidative damage. Project 4 was added to the original POI in September, 2007, to identify rate-limiting steps in the BER of lesions in nucleosomes, and to investigate mechanisms that cells use to circumvent these limits. Studies to date indicate that the human bifunctional DNA glycosylase hNTHI can excise oxidative lesions from nucleosomal DNA without irreversibly disrupting the nucleosome, and that spontaneous, transient partial unwrapping of DNA from the histone octamer facilitates access to sterically occluded lesions. With these results as a foundation, the goals for this new proposal are to test HYPOTHESIS 1, that during BER of lesions in nucleosomes, there is a stepwise hand-off of substrates from one enzyme to the next, facilitated by BER accessory factors. In the course of testing this hypothesis, we will investigate changes in nucleosome structure that accompany individual steps in BER, These structural analyses will help guide future studies of histone chaperones and chromatin remodeling agents that facilitate BER in vivo. We also will test HYPOTHESIS 2, that histone primary sequence variants associated with either transcriptionally active or inactive chromatin influence the capacity of selected human DNA glycosylases to repair oxidative damages in nucleosomes. Information from Project 2 will help elucidate the molecular basis for interactions between hNTHI and lesion-containing nucleosomes. Information and reagents provided by Core A and Projects 1 and 3 will enable us to test HYPOTHESIS 3, that DNA polymerase p (Pol P) variants with demonstrated oncogenic potential differ from wild type Pol p in BER of lesions in nucleosomes. We also will determine If, in the context of nucleosomal templates, Pol p (or oncogenic variants of Pol P) can bind or recruit hRadSI. Core B will provide critical reagents for all of the studies in Project 4.

Public Health Relevance

The information generated by the proposed studies will advance our understanding of the carcinogic consequences of damages arising from endogenous metabolism and radiafion exposure. Additionally, radiation threatment is widely used in cancer therapy. The proposed studies will advance our understanding of factors that affect radiafion sensitivity of cells and modulation of such factors could help maximize therapeutic gain during radiotherapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA098993-10
Application #
8725061
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
DUNS #
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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