This proposal examines the role of mitogen-activated protein (MAP) kinase pathways in nucleotide excision repair (NER). Studies focus on c-Jun-NH2-terminal kinase (JNK)-mediated activation of the transcription/repair factor TFIIH. NER is the predominant mechanism in eukaryotic cells for repair of ultraviolet (UV) or cis-platinum-induced DNA damage. Mutations in TFIIH, result in several inheritable diseases, Xeroderma pigmentosa (XP), Cockaynes's Syndrome (CS) and Trichothiodystrophy (TTD), marked by defects in global or transcription-coupled repair, and in XP, a predisposition to skin cancers. (Disruptions in JNK result in decreased NER and increased cytotoxicity following DNA damage. Links between MAPKs and NER will be developed through the following experimental aims: 1. Measure cytotoxicity and repair of UV- and cis-platinum- induced DNA damage in cells deficient for MAP kinase signaling. 2. Determine specific TFIIH subunits/sites phosphorylated by activated MAP kinases in vivo and in vitro following exposure of cells to UV or cis-platinum. 3. Assess the effects of MAP kinase-mediated phosphorylation on TFIIH activities.
Aim one will be accomplished through the use of MTS Cytotox (Promega) and quantitative PCR assays in HeLa and JNK-/- cells.
Aim 2 will be examined by orthophosphate labeling of whole cells or phosphatase treatment of immunoprecipitated TFIIH from cells expressing dominant negative MAPK or deleted for JNK isozymes (JNK-/-). Phosphorylation of TFIIH subunits by MAP kinases, will be determined through in vitro kinase assays using GST/His fusion proteins of TFIIH and activated MAP kinases. Phosphorylation domains/sites will be explored through phosphoamino acid analysis and confirmed with deletion and site specific mutants.
Aim 3 will be examined using assays specific for subunit activities and complex activity TFIIH in transcription and NER.