Transcriptional inhibition by an oxidized abasic site in DNA.
2-Deoxyribonolactone (dL) is an oxidized abasic site in DNA that can be induced by gamma-radiolysis, ultraviolet irradiation, and numerous antitumor drugs. Although this lesion is incised by AP endonucleases, suggesting a base-excision repair mechanism for dL removal, subsequent excision and repair synthesis by DNA polymerase beta is inhibited due to accumulation of a protein-DNA cross-link. This raises the possibility that additional repair pathways might be required to eliminate dL from the genome. Transcription-coupled repair (TCR) is a pathway of excision repair specific to DNA lesions present in transcribed strands of expressed genes. A current model proposes that transcription arrest at the site of DNA damage is required to initiate TCR. In support of this model, a strong correlation between transcription arrest by a lesion in vitro and TCR of the lesion in vivo has been found in most cases analyzed. To assess whether dL might be subject to TCR, we have studied the behavior of bacteriophage T3 and T7 RNA polymerases (T3RNAP, T7RNAP) and of mammalian RNA polymerase II (RNAPII) when they encounter a dL lesion or its "caged" precursor located either in the transcribed or in the nontranscribed strand of template DNA. DNA plasmids containing a specifically located dL downstream of the T3, T7 promoter or the Adenovirus major late promoter were constructed and used for in vitro transcription with purified proteins. We found that both dL and its caged precursor located in the transcribed strand represented a complete block to transcription by T3- and T7RNAP. Similarly, they caused more than 90% arrest when transcription was carried out with mammalian RNAPII. Furthermore, RNAPII complexes arrested at dL were subject to the transcript cleavage reaction mediated by elongation factor TFIIS, indicating that these complexes were stable. A dL in the nontranscribed strand did not block either polymerase.
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, USA.
Yingli
Y
Wang
Terry L
TL
Sheppard
Silvia
S
Tornaletti
Lauren S
LS
Maeda
Philip C
PC
Hanawalt
D000818:Animals; D017903:Bacteriophage T3 / Q000201:enzymology; D017123:Bacteriophage T7 / Q000201:enzymology; D004247:DNA / Q000187:drug effects* / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000235:genetics; D051381:Rats; D013400:Sugar Acids / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics
D013400:Sugar Acids; C056405:2,4,5-trihydroxypentanoic acid gamma-lactone; D004247:DNA; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases