The Escherichia coli uvr ABC system catalyzes the incision of damaged DNA. Having available reagent quantities of homogeneous UurB and UvrC proteins permits a detailed examination of the individual partial reactions leading to the dual incision stage of nucleotide excision repair. The pre-incision steps can be subdivided into a number of partial reactions which include: (a) UvrA dimerization stimulated by ATP binding, (b) UvrA-nucleoprotein formation at both damaged and undamaged sites, (c) the accompanying topological unwinding stimulated by ATP binding, (d) the participation of the UvrB cryptic ATPase in the UvrAB catalyzed strand displacement reaction and finally (e) dual incision catalyzed by the presence of UvrC. The incision mechanisms precede the multi-nucleoprotein complex requiring coordinated excision reactions catalyzed by UvrD, DNA polymerase I and polynucleotide ligase. In the principal direction for the proposed studies we will attempt to associate the anatomy, or structure of the respective uvr A and B genes to the catalytic and protein properties of the related gene product proteins. The focal points and role of ATP in the individual processes will also be addressed. The protein sequences, or domains, of interest include putative ATP binding regions, sites sensitive to a protease specific for the Ada protein of E. coli potential DNA binding sites and to a limited extent the """"""""zinc finger""""""""-like sites. These sites will be engineered by oligonucleotide-directed and deletion mutants, the individual clones sequenced and over-expressed in suitable expression vectors and the catalytic and protein properties of the mutant and """"""""wild type"""""""" proteins examined for their enzymatic phenotypes in the respective pre-, post- and incision steps. The biological role and biochemical nature of UvrB proteolysis in regulation of repair will be further investigated by genetic, structural and catalytic methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM022846-24
Application #
2770895
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-09-01
Project End
2000-08-31
Budget Start
1998-09-01
Budget End
2000-08-31
Support Year
24
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Baccarelli, A; Calista, D; Minghetti, P et al. (2004) XPD gene polymorphism and host characteristics in the association with cutaneous malignant melanoma risk. Br J Cancer 90:497-502
Qiao, Yawei; Spitz, Margaret R; Shen, Hongbing et al. (2002) Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes. Carcinogenesis 23:295-9
Qiao, Yawei; Spitz, Margaret R; Guo, Zhaozheng et al. (2002) Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes. Mutat Res 509:165-74
Landi, Maria Teresa; Baccarelli, Andrea; Tarone, Robert E et al. (2002) DNA repair, dysplastic nevi, and sunlight sensitivity in the development of cutaneous malignant melanoma. J Natl Cancer Inst 94:94-101
Grossman, L (2001) Nucleotide excision repair: Dick Setlow: how he influenced my scientific life. Environ Mol Mutagen 38:144-52
Hildebrand, E L; Grossman, L (1999) Oligomerization of the UvrB nucleotide excision repair protein of Escherichia coli. J Biol Chem 274:27885-90
Kovalsky, O I; Lin, C G; Grossman, L (1998) Selection of monoclonal antibodies for probing of functional intermediates in incision of UV-irradiated DNA by Uvr(A)BC endonuclease from Escherichia coli. Biochim Biophys Acta 1397:91-101
Kovalsky, O I; Grossman, L (1998) Accessibility of epitopes on UvrB protein in intermediates generated during incision of UV-irradiated DNA by the Escherichia coli Uvr(A)BC endonuclease. J Biol Chem 273:21009-14
Hildebrand, E L; Grossman, L (1998) Introduction of a tryptophan reporter group into the ATP binding motif of the Escherichia coli UvrB protein for the study of nucleotide binding and conformational dynamics. J Biol Chem 273:7818-27
ClGL; Kovalsky, O; Grossman, L (1998) Transcription coupled nucleotide excision repair by isolated Escherichia coli membrane-associated nucleoids. Nucleic Acids Res 26:1466-72

Showing the most recent 10 out of 36 publications