We have used in vitro and in vivo RNA polymerase II assays to measure the level of RNA synthesis in cell free extracts, in chromatin in permeabilized cells, or in whole, intact cells. We find reduced RNA polymerase II transcription in Cockayne syndrome (CS) cells compared to normal human cells. In contrast, the transcription levels for RNA polymerases I and III are normal in CS cell extracts. By fractionation of whole cell extracts we identify one fraction which is repair defective in the CS cells. The reduced transcription by RNA polymerase II can be corrected by mixing specific fractions of extracts from CS and normal cells. The transcription defect in CS cells can be corrected by the addition of normal cell extract or by transfection of CS cells with a plasmid carrying a copy of the normal CS gene. We find that both whole cell extracts and reconstituted fractions of XP-B and XP-D cells show reduced levels of transcription while extracts of XP-A, X-PC, XP-F and XP-G have normal RNA polymerase II transcription. This suggests that the human DNA repair genes XP-B and XP-D are directly involved in transcription, whereas the repair genes XP-A, XP-C, XP-G and XP-F do not appear to play a role in transcription. We have used in vitro and in vivo RNA polymerase II assays to measure the level of RNA synthesis in cell free extracts, in chromatin of permeabilized cells, or in intact cells. Current data show a reduced leve of RNA polymerase II transcription in Werner syndrome cells compared to normal cells. We find that there is a significant conversion of RNAPII from the unphosphorylated form to phosphorylated forms after UVC irradiation. Further characterization of this UVC response revealed detectable phosphorylation after irradiation at a fluency of 20 J/m2 that reached a maximal level at a fluency of 40 J/m2. The CTD kinase activity seemed to briefly precede the appearance of phosphorylated RNAPII. After treatment with increasing doses of UVC radiation, hepta-4 kinase activity was detected at a fluency of 20 J/m2 and reached a maximum level at a fluency of 40 J/m2.
Showing the most recent 10 out of 14 publications
