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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Program Projects (P01)
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Special Emphasis Panel (ZCA1)
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University of Vermont & St Agric College
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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
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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