The applicant's long-term research objectives are to elucidate biochemically the mechanisms by which a human cell safeguards its genetic integrity. The short term plan focuses on the nucleotide excision repair (NER) pathway, whose malfunction leads to the hereditary disease xeroderma pigmentosum, which is characterized by increased frequency to skin cancers and symptoms associated with defective neural development. This research proposal outlines studies on the multi-subunit protein complex transcription-repair factor IIH (TFIIH), which plays a key role in NER and associates with the cyclin-dependent kinase (CDK)-activating kinase (CAK). Recently, the applicant has isolated two novel human TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3, p62, p44, p41, and p34. ERCC2/CAK was isolated as a complex composed of the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 protein. Combined action of TFIIH* and ERCC2/CAK efficiently supports RNA polymerase II-catalyzed transcription, and each of these complexes is active in supporting NER reactions. In this FIRST grant period, the applicant proposes: i) to further investigate the mechanisms underlying significance of its association with CAK, and ii) to investigate the mechanisms underlying gap-filling DNA resynthesis during NER.
Specific aims are to: 1) determine the function of the CAK-TFIIH association in DNA repair, 2) assess the role of CAK-TFIIH kinase activity in coordinating NER and the p53-dependent checkpoint pathway, 3) determine the role of ERCC2/CAK and TFIIH* in damage recognition and assembly of an ATP-dependent preincision complex, and 4) investigate mechanisms that couple the post-incision reaction and gap-filling DNA synthesis during NER.
