The Protein and Biochemistry Core will express and purify proteins for all members of the Program Project. In addition. Core B will perform quantitative analyses of protein-DNA interactions and characterize the kinetic properties of DNA repair proteins using high-throughput fluorimetric assays.
Specific Aim 1 : (A) To optimize the expression and purification of DNA repair proteins Core B will produce mg amounts of soluble proteins for Projects 1, 2, 3, and 4. We will express the proteins in different E. coli strains using protocols and methods that have proven successful in the past four years in our laboratory, such as autoinduction. We will continue to use limited proteolysis in conjunction with bioinformatics to delineate functional domains and express smaller fragments, if the full-length protein construct fails to overexpress. (B) To optimize the solubility and stability of proteins and complexes to be used in crystallization experiments, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Crystallization trials using commercial kits will be set up using a robotic workstation.
Specific Aim 2 : To perform rapid quantitative analyses of protein-DNA interactions and steady-state enzyme kinetic properties of wild-type and mutant DNA repair enzymes generated in this Program Project, using high-throughput fluorimetric assays

Public Health Relevance

Core B will play an integral role in providing purified proteins and performing quantitative analyses of the properties of DNA glycosylases and recombination proteins. We expect the results of the studies proposed with this Program Project to advance our understanding of how DNA repair protein variants contribute to cancer susceptibility and as well provide useful targets for cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA098993-10
Application #
8725063
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
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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