The Expression, Characterization, and Crystallization (ECC) Core will optimize protein expression for Program Project members, as well as design and engineer site-directed or deletion mutants for proteins targeted for crystallography. In addition, the ECC Core will perform quantitative analyses of protein-DNA interactions and complex stability. We will use the crystallization robot in the Crystallographic Facility to set up crystallization trials of enzymes in complex with their DNA substrates.
Aim 1 To optimize the expression of proteins of interest. We will optimize protein expression in E. coli cells first, by varying the cell strains and other factors, such as temperature, or the incubation medium. The protein constructs that do not express well in E. coli or are not active will be expressed in insect cells. We will use the Gateway system to easily switch from the bacterial to the eukaryotic expression system.
Aim 2 : To design and engineer site directed or deletion mutants of the proteins targeted for crystallography. We will make use of Bioinformatics Core A to choose the mutation to make in each glycosylase or recombinase that would potentially affect its activity.
Aim 3 : (A) To perform rapid quantitative analyses of enzyme activity using high-throughput fluorimetric assays and to optimize the solubility and stability of proteins and complexes to be used in crystallization experiments. To this end, we will use a combination of limited proteolysis, prediction of disordered regions and dynamic light scattering to delineate smaller domains that retain the ability to bind and cleave DNA substrates. We will use both commercially available crystallization screens and custom-designed incomplete factorial screens to search for crystallization conditions for the recombinase and glycosylase complexes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA098993-04
Application #
7500165
Study Section
Subcommittee G - Education (NCI)
Project Start
Project End
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
4
Fiscal Year
2007
Total Cost
$280,428
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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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
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
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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