The two main goals are to: (1) Understand how mutations in the ERCC2 DNA helicase produce two different diseases, cancer-prone xeroderma pigmentosum (XP) and developmentally abnormal trichothiodystophy (TTD), and (2 Define structure-function relationships of ERCC2 as a model helicase. ERCC2 is a component of the TFIIH transcription-repair complex, and the unwinding activity of ERCC2 is required for nucleotide excision repair (NER) but not transcription. In XP group D (XP-D), mutations in the ERCC2 gene generally confer high ultraviolet (UV) sensitivity and map to regions of the protein containing highly conserved domains responsible for unwinding activity. In contrast, TTD mutations also confer UV sensitivity but map to the C-terminus outside the helicase domains and may cause subtle transcription defects by affecting the stability of the TFIIH complex. Using a CHO hamster-cell system, the general hypothesis to be tested is that XP-D mutations produce ERCC2 proteins that lack unwinding activity while TTD mutations result in weak association of ERCC2 with other proteins in the TFIIH complex. This concept will be tested by determining whether XP mutations, but not TTD mutations, can produce a dominant phenotype, which manifests as UV sensitivity, when they are overexpressed in wild-type hamster cells. To examine the idea that TTD results from a weak association of ERCC2 with partner proteins, it will be determined whether UV resistance can be restored by overexpressing TTD mutations in the absence of normal protein. The main hypothesis will also be tested by purifyin normal and mutant ERCC2 from an overexpression system, and then performing detailed characterization of purified proteins in order to correlate functiona changes with mutations in specific domains. These studies will determine whether and why XP-D proteins lose unwinding activity and whether TTD proteins retain unwinding activity. A novel approach using intragenic suppressor mutations will help to elucidate relationships among the helicase domains and to define the function(s) associated with each domain. These mechanistic insights will guide future structural studies on ERCC2.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA052679-10
Application #
6172427
Study Section
Radiation Study Section (RAD)
Program Officer
Pelroy, Richard
Project Start
1991-05-20
Project End
2003-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
10
Fiscal Year
2000
Total Cost
$436,501
Indirect Cost
Name
Lawrence Livermore National Laboratory
Department
Biology
Type
Organized Research Units
DUNS #
827171463
City
Livermore
State
CA
Country
United States
Zip Code
94550
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Kadkhodayan, S; Salazar, E P; Ramsey, M J et al. (1997) Molecular analysis of ERCC2 mutations in the repair deficient hamster mutants UVL-1 and V-H1. Mutat Res 385:47-57
Kadkhodayan, S; Salazar, E P; Lamerdin, J E et al. (1996) Construction of a functional cDNA clone of the hamster ERCC2 DNA repair and transcription gene. Somat Cell Mol Genet 22:453-60
Takayama, K; Salazar, E P; Broughton, B C et al. (1996) Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy. Am J Hum Genet 58:263-70
Kirchner, J M; Salazar, E P; Lamerdin, J E et al. (1994) Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene. Genomics 23:592-9
Weber, C A; Kirchner, J M; Salazar, E P et al. (1994) Molecular analysis of CXPD mutations in the repair-deficient hamster mutants UV5 and UVL-13. Mutat Res 324:147-52