The Protein Expression and Cell Culture Core will provide expertise and support for all Program Project investigators. The Core will express and purify proteins for Projects 2, 3, and 4. In addition, Core B will also generate mammalian cell lines and provide cell culture services for all projects. The Core Director, Dr. Doubli, has extensive experience in protein expression and purification. She will continue to oversee the protein production aspect of the Core and directly supervise the staff in charge of protein expression, purification, optimization and preliminary crystallization screens. Dr. Sweasy, the Co-Director, has extensive experience in the construction of mammalian cell lines and in the characterization of DNA repair genes in tissue culture cells. Core B will produce mg amounts of soluble proteins for Projects 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 in our laboratory, such as autoinduction. The Core will also optimize the solubility and stability of proteins and complexes to be used in biochemical assays, crystallization experiments and single molecule studies, by characterizing solvent effects on protein aggregation properties using dynamic light scattering and analytical gel filtration. Initial crystallization trials using commercial kits will be set up using a robotic workstation. Core B will also produce mammalian cell lines for all projects. The Core will screen the resulting cell lines for expression of the protein of interest. Mammalian proteins of interest will be expressed in HEK293 cells for biochemical experiments. The Core will also assist members of the Program with propagation of their cell lines and experiment design. The Core is well equipped to express and purify proteins. The technician in charge of protein production has worked in Core B for the last 8 years and is highly qualified. Core B is also well equipped for tissue culture work, having the necessary incubators, hoods, microscope, centrifuge, and storage facilities. A full-time technician with prior experience in mammalian cell culture techniques is in charge of the cell culture work. The services provided by Core B are essential for each Project and for the success of the Program Project as a whole. The Protein Expression and Cell Culture services are unique on the University of Vermont campus and do not duplicate any University core facilities already available to our research group.

Public Health Relevance

Core B will play an integral role in providing purified proteins and mammalian cell culture lines to all Projects. We expect the results of the studies proposed within this Program Project to advance our understanding of how DNA repair protein variants contribute to cancer susceptibility.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
2P01CA098993-11A1
Application #
9209393
Study Section
Special Emphasis Panel (ZCA1-RPRB-F (O1))
Project Start
2004-09-03
Project End
2022-04-30
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
11
Fiscal Year
2017
Total Cost
$258,400
Indirect Cost
$92,493
Name
University of Vermont & St Agric College
Department
Type
Domestic Higher Education
DUNS #
066811191
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
Cannan, Wendy J; Pederson, David S (2016) Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin. J Cell Physiol 231:3-14

Showing the most recent 10 out of 64 publications