Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template.
A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcription-coupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SII (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SII induces nascent transcript cleavage by RNA polymerase II stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.
Donahue
B A
BA
Department of Biological Sciences, Stanford University, CA 94305-5020.
Yin
S
S
Taylor
J S
JS
Reines
D
D
Hanawalt
P C
PC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011740:Pyrimidine Dimers / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D035165:Transcription Factors, General; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D012333:RNA, Messenger; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II; D004255:Deoxyribodipyrimidine Photo-Lyase