9630773 Labhart A detailed analysis of transcription factor phosphorylation resulting from activation of a signaling pathway is essential for a complete understanding of signal transduction regulation of cell proliferation and differentiation. DNA-dependent protein kinase (DNA-PK) is a moderately abundant nuclear kinase that is activated by binding to double-stranded DNA ends and DNA structures bearing single-to double strand transitions. DNA-PK has been shown to have a role in repair of double-stranded DNA breaks, but its precise function and its in vivo substrates are not known. DNA-PK has also been shown to phosphorylate many transcription factors that are bound to the activating DNA molecule. Yet, the physiological role of DNA-PK in transcription is not known. DNA-PK strongly inhibits transcription by Xenopus RNA polymerase I in an in vitro system. RNA polymerase I is the enzyme that synthesizes the large ribosomal RNAs that -- after processing of a precursor molecule -- are incorporated in the ribosomes. DNA-PK does not affect transcription by the other nuclear RNA polymerases, class II and III.. Since DNA-PK is a central player in this project, it is also proposed to purify Xenopus DNA-PK and to initiate the molecular characterization of this enzyme. The investigator will address the question about the in vivo significance of the repression of ribosomal gene transcription by DNA-PK. He will examine whether there is a correlation between the activity and subcellular localization of DNA-PK and the rate of synthesis of ribosomal RNA during Xenopus oogenesis and embryogenesis. If DNA-PK negatively regulates ribosomal gene transcription in the cell, high DNA-PK activity and nucleolar localization would be expected to correlate with a low rate of ribosomal RNA synthesis. Furthermore, he will attempt to inhibit the expression and/or activity of the endogenous DNA-PK and investigate any effects on ribosomal gene transcription, oogenesis and embryogenesis. The elucidation of the mechan ism by which DNA-PK represses ribosomal gene transcription promises to advance the knowledge about this nuclear enzyme and will help to eventually identify its in vivo significance and its role in transcription. ***
