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)
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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Cannan, Wendy J; Tsang, Betty P; Wallace, Susan S et al. (2014) Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages. J Biol Chem 289:19881-93
Wallace, Susan S (2014) Base excision repair: a critical player in many games. DNA Repair (Amst) 19:14-26
Nelson, Shane R; Dunn, Andrew R; Kathe, Scott D et al. (2014) Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases. Proc Natl Acad Sci U S A 111:E2091-9
Lubula, Mulu Y; Poplawaski, Amanda; Glass, Karen C (2014) Crystallization and preliminary X-ray diffraction analysis of the BRPF1 bromodomain in complex with its H2AK5ac and H4K12ac histone-peptide ligands. Acta Crystallogr F Struct Biol Commun 70:1389-93
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
Sjolund, Ashley; Nemec, Antonia A; Paquet, Nicolas et al. (2014) A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation. PLoS Genet 10:e1004753
Lee, Andrea J; Warshaw, David M; Wallace, Susan S (2014) Insights into the glycosylase search for damage from single-molecule fluorescence microscopy. DNA Repair (Amst) 20:23-31
Prakash, Aishwarya; Eckenroth, Brian E; Averill, April M et al. (2013) Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases. DNA Repair (Amst) 12:1062-71
Liu, Minmin; Doublie, Sylvie; Wallace, Susan S (2013) Neil3, the final frontier for the DNA glycosylases that recognize oxidative damage. Mutat Res 743-744:4-11
Odell, Ian D; Wallace, Susan S; Pederson, David S (2013) Rules of engagement for base excision repair in chromatin. J Cell Physiol 228:258-66

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