The central hypothesis of this Program Project is that defects in base excision repair (BER) drive human carcinogenesis and affect responses to cancer treatments. To test this hypothesis, we are using our strengths in fundamental biochemistry, molecular biology, structural biology and biophysics to examine human genetic variation in the BER enzymes. Our program is informed and driven by the identification of germline and tumor- associated enzyme variants that may alter the DNA repair capacity of human BER enzymes. The single- molecule approaches proposed in Project 4 will provide insights into how the human DNA glycosylases and the downstream enzymes in BER search for their targets in a sea of undamaged DNA as well as in a chromatin milieu. We hypothesize that, as we have shown for their bacterial homologs, the human DNA glycosylases, which remove oxidized bases, scan the DNA in a rotational manner employing an amino acid wedge to search for base damage. We further postulate that the downstream enzymes search for the product- bound prior enzyme in the pathway and that DNA compacted into nucleosomes may reduce BER enzyme diffusion rates. These hypotheses will be tested by: Elucidating the real-time DNA damage search behavior of the human DNA glycosylases and their variants (Aim 1); elucidating the spatial relationships and interactions between enzymes in the BER pathway relative to the site of DNA damage (Aim 2); and determining the diffusive behavior of the BER enzymes in the context of chromatin (Aim 3). To do this, we will examine the diffusive properties of the BER enzymes on both undamaged and site-specifically damaged DNA and on chromatin as well as on DNA or chromatin bound by the prior enzyme in the pathway. We also will determine if the diffusion of glycosylases is affected by mutations in putative reading head amino acids or by germline and tumor-associated variants that potentially affect the BER search process.

Public Health Relevance

Aberrant DNA repair is a key cause of genomic instability leading to cancer and tumor progression. The results of the studies proposed in Project 4 will provide mechanistic insights into how mutations in DNA repair genes in the normal population and in tumors contribute to altered DNA repair capacity and enhance our understanding of carcinogenesis. Information from these studies should also lead to novel therapeutic strategies for tumors containing mutations in DNA repair genes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
2P01CA098993-11A1
Application #
9209398
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
$366,578
Indirect Cost
$131,215
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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