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.
