Transcription blockage by bulky end termini at single-strand breaks in the DNA template: differential effects of 5' and 3' adducts.
RNA polymerases from phage-infected bacteria and mammalian cells have been shown to bypass single-strand breaks (SSBs) with a single-nucleotide gap in the template DNA strand during transcription elongation; however, the SSB bypass efficiency varies significantly depending upon the backbone end chemistries at the break. Using a reconstituted T7 phage transcription system (T7 RNAP) and RNA polymerase II (RNAPII) in HeLa cell nuclear extracts, we observe a slight reduction in the level of transcription arrest at SSBs with no gap as compared to those with a single-nucleotide gap. We have shown that biotin and carbon-chain moieties linked to the 3' side, and in select cases the 5' side, of an SSB in the template strand strongly increase the level of transcription arrest when compared to unmodified SSBs. We also find that a small carbon-chain moiety linked to the upstream side of an SSB aids transcriptional bypass of SSBs for both T7 RNAP and RNAP II. Analysis of transcription across SSBs flanked by bulky 3' adducts reveals the ability of 3' end chemistries to arrest T7 RNAP in a size-dependent manner. T7 RNAP is also completely arrested when 3' adducts or 3'-phosphate groups are placed opposite 5'-phosphate groups at an SSB. We have also observed that a biotinylated thymine in the template strand (without a break) does not pose a strong block to transcription. Taken together, these results emphasize the importance of the size of 3', but usually not 5', end chemistries in arresting transcription at SSBs, substantiating the notion that bulky 3' lesions (e.g., topoisomerase cleavable complexes, 3'-phosphoglycolates, and 3'-unsaturated aldehydes) pose very strong blocks to transcribing RNA polymerases. These findings have implications for the processing of DNA damage through SSB intermediates and the mechanism of SSB bypass by T7 RNAP and mammalian RNAPII.
Department of Biology, Stanford University, Stanford, California 94305, United States.
Alexander J
AJ
Neil
Boris P
BP
Belotserkovskii
Philip C
PC
Hanawalt
D017123:Bacteriophage T7 / Q000378:metabolism; D018736:DNA Adducts / Q000494:pharmacology*; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D012319:RNA Polymerase II / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D018736:DNA Adducts; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases