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.
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.
|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|
|Marsden, Carolyn G; Dragon, Julie A; Wallace, Susan S et al. (2017) Base Excision Repair Variants in Cancer. Methods Enzymol 591:119-157|
|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|
|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|
|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|
|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|
|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|
|Prakash, Aishwarya; Moharana, Kedar; Wallace, Susan S et al. (2017) Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase. Nucleic Acids Res 45:2897-2909|
|Lee, Andrea J; Wallace, Susan S (2017) Hide and seek: How do DNA glycosylases locate oxidatively damaged DNA bases amidst a sea of undamaged bases? Free Radic Biol Med 107:170-178|
|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|
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