Nucleotide excision repair is a DNA repair mechanism that involves the removal of damaged nucleotides from double-stranded DNA followed by closure of the resulting gap. In E. coli, UVRABC nuclease, encoded by the uvrA, uvrB and uvrC genes, catalyzes the removal of UV-induced pyrimidine dimers (and presumably other carcinogen-induced base adducts which distort the DNA double helix) by hydrolyzing the 8th phosphodiester bond 5' and the 4th or 5th phosphodiester bond 3' to the modified nucleotides and removing the resulting 12-13 nucleotide long single-stranded DNA fragment. This project will further characterize UVRABC nuclease and the nucleotide excision repair mechanism in E. coli. Subunit composition of UVRABC nuclease will be determined by sedimentation in glycerol gradients and by gel filtration chromatography. Temperature sensitive and deletion mutants of the uvrA, uvrB and uvrC proteins will be isolated; purified mutant proteins will be used to define the functional domains of the subunits and the role of each in the two incision reactions. The sequences of the uvr proteins will be obtained by DNA sequence analysis and the secondary structures predicted. DNA fragments containing various base adducts will be prepared. The incision sites relative to these adducts will be determined by incubating terminally labeled fragments with UVRABC nuclease and analyzing the reaction products on DNA sequencing gels. Assays measuring the binding, incision, and excision steps of the reaction will be developed to determine the thermodynamic and kinetic parameters governing the interaction of the substrate with the enzyme and its subunits. The chemical groups of the substrate in contact with the enzyme will be identified by alkylation protection and interference methods using a substrate of defined sequence carrying a single pyrimidine dimer. Nucleotide excision repair will be reconstituted in vitro using UVRABC nuclease, DNA polymerase I and E. coli DNA ligase. This system will be used to investigate the origin of the short and long repair patches observed in vivo and the involvement of recA protein in long patch repair.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032833-04
Application #
3281979
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-12-01
Project End
1992-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Kemp, Michael G (2017) Crosstalk Between Apoptosis and Autophagy: Environmental Genotoxins, Infection, and Innate Immunity. J Cell Death 9:1179670716685085
Kemp, Michael G; Hu, Jinchuan (2017) PostExcision Events in Human Nucleotide Excision Repair. Photochem Photobiol 93:178-191
Song, Jimyeong; Kemp, Michael G; Choi, Jun-Hyuk (2017) Detection of the Excised, Damage-containing Oligonucleotide Products of Nucleotide Excision Repair in Human Cells. Photochem Photobiol 93:192-198
Canturk, Fazile; Karaman, Muhammet; Selby, Christopher P et al. (2016) Nucleotide excision repair by dual incisions in plants. Proc Natl Acad Sci U S A 113:4706-10
Adar, Sheera; Hu, Jinchuan; Lieb, Jason D et al. (2016) Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis. Proc Natl Acad Sci U S A 113:E2124-33
Kemp, Michael G; Sancar, Aziz (2016) ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress. J Biol Chem 291:9330-42
Gaddameedhi, Shobhan; Selby, Christopher P; Kemp, Michael G et al. (2015) The circadian clock controls sunburn apoptosis and erythema in mouse skin. J Invest Dermatol 135:1119-1127
Kemp, Michael G; Lindsey-Boltz, Laura A; Sancar, Aziz (2015) UV Light Potentiates STING (Stimulator of Interferon Genes)-dependent Innate Immune Signaling through Deregulation of ULK1 (Unc51-like Kinase 1). J Biol Chem 290:12184-94
Lindsey-Boltz, Laura A; Kemp, Michael G; Capp, Christopher et al. (2015) RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling. Cell Cycle 14:99-108
Choi, Jun-Hyuk; Kim, So-Young; Kim, Sook-Kyung et al. (2015) An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS. J Biol Chem 290:28812-21

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