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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Intramural Research (Z01)
Project #
1Z01AG000731-01
Application #
2447740
Study Section
Special Emphasis Panel (LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1996
Total Cost
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Thorslund, Tina; von Kobbe, Cayetano; Harrigan, Jeanine A et al. (2005) Cooperation of the Cockayne syndrome group B protein and poly(ADP-ribose) polymerase 1 in the response to oxidative stress. Mol Cell Biol 25:7625-36
Christiansen, Mette; Thorslund, Tina; Jochimsen, Bjarne et al. (2005) The Cockayne syndrome group B protein is a functional dimer. FEBS J 272:4306-14
Tuo, Jingsheng; Jaruga, Pawel; Rodriguez, Henry et al. (2003) Primary fibroblasts of Cockayne syndrome patients are defective in cellular repair of 8-hydroxyguanine and 8-hydroxyadenine resulting from oxidative stress. FASEB J 17:668-74
Licht, Cecilie Loe; Stevnsner, Tinna; Bohr, Vilhelm A (2003) Cockayne syndrome group B cellular and biochemical functions. Am J Hum Genet 73:1217-39
Kyng, Kasper J; May, Alfred; Brosh Jr, Robert M et al. (2003) The transcriptional response after oxidative stress is defective in Cockayne syndrome group B cells. Oncogene 22:1135-49
Christiansen, Mette; Stevnsner, Tinna; Modin, Charlotte et al. (2003) Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein. Nucleic Acids Res 31:963-73
Bohr, Vilhelm A (2002) Human premature aging syndromes and genomic instability. Mech Ageing Dev 123:987-93
Tuo, Jingsheng; Chen, Catheryne; Zeng, Xianmin et al. (2002) Functional crosstalk between hOgg1 and the helicase domain of Cockayne syndrome group B protein. DNA Repair (Amst) 1:913-27
Selzer, Rebecca R; Nyaga, Simon; Tuo, Jingsheng et al. (2002) Differential requirement for the ATPase domain of the Cockayne syndrome group B gene in the processing of UV-induced DNA damage and 8-oxoguanine lesions in human cells. Nucleic Acids Res 30:782-93
Bohr, V A (2002) DNA damage and its processing. relation to human disease. J Inherit Metab Dis 25:215-22

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