Effects of the bacterial transcription-repair coupling factor during transcription of DNA containing non-bulky lesions.
Transcription-coupled DNA repair is a mechanism by which bulky DNA lesions that block transcription by RNA polymerase are prioritised for removal by the nucleotide excision repair apparatus. The trigger is thought to be the presence of an irreversibly blocked transcription complex, which is recognised by a transcription-repair coupling factor. Many common DNA lesions do not block transcription, but are bypassed with varying degrees of efficiency and with potentially mutagenic effects on the RNA transcripts that are produced. The effect of the bacterial transcription-repair coupling factor, Mfd, at such lesions is not known: it has been suggested that Mfd may promote mutagenesis by increasing the efficiency with which RNA polymerase bypasses non-bulky lesions, but it has also been reported that 8-oxoguanine, a major product of oxidative DNA damage that is efficiently bypassed by RNA polymerase, is subject to Mfd-dependent transcription-coupled repair in Escherichia coli. We have investigated the effect of Mfd during transcription of templates containing 8-oxoguanine, and various other non-bulky lesions. We show that an 8-oxoguanine in the template strand induces a transient pause in transcription, and that Mfd neither increases nor decreases the efficiency with which RNA polymerase bypasses the lesion. We also show that Mfd can displace a transcription complex stalled at a single strand nick, and that it decreases the efficiency with which RNA polymerase bypasses an abasic site. These activities are not affected by transcription rate, as similar results were obtained using "fast" and "slow" mutant RNA polymerases. Our findings suggest that 8-oxoguanine is unlikely to be directly targeted by the transcription-coupled repair pathway, and identify a potential role for Mfd in reducing the level of transcriptional mutagenesis caused by abasic sites.
Smith
Abigail J
AJ
DNA-protein Interactions Unit, Department of Biochemistry, University of Bristol, Bristol, UK.
Savery
Nigel J
NJ
D001426:Bacterial Proteins / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D004249:DNA Damage; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014498:Uracil / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D050505:Mutant Proteins; D014157:Transcription Factors; C072103:transcription repair coupling factor protein, Bacteria; C024829:8-hydroxyguanine; D014498:Uracil; D006147:Guanine; D012321:DNA-Directed RNA Polymerases