Transcription of DNA containing the 5-guanidino-4-nitroimidazole lesion by human RNA polymerase II and bacteriophage T7 RNA polymerase.
Damage in transcribed DNA presents a challenge to the cell because it can partially or completely block the progression of an RNA polymerase, interfering with transcription and compromising gene expression. While blockage of RNA polymerase progression is thought to trigger the recruitment of transcription-coupled DNA repair (TCR), bypass of the lesion can also occur, either error-prone or error-free. Error-prone transcription is often referred to as transcriptional mutagenesis (TM). Elucidating why some lesions pose blocks to transcription elongation while others do not remains a challenging problem. As part of an effort to understand this, we studied transcription past a 5-guanidino-4-nitroimidazole (NI) lesion, using two structurally different RNA polymerases, human RNA polymerase II (hRNAPII) and bacteriophage T7 RNA polymerase (T7RNAP). The NI damage results from the oxidation of guanine in DNA by peroxynitrite, a well known, biologically important oxidant. It is of structural interest because it is a ring-opened and conformationally flexible guanine lesion. Our results show that NI acts as a partial block to T7RNAP while posing a major block to hRNAPII, which has a more constrained active site than T7RNAP. Lesion bypass by T7RNAP induces base misincorporations and deletions opposite the lesion (C>A>-1 deletion >G >>> U), but hRNAPII exhibits error-free transcription although lesion bypass is a rare event. We employed molecular modeling methods to explain the observed blockage or bypass accompanied by nucleotide incorporation opposite the lesion. The results of the modeling studies indicate that NI's multiple hydrogen-bonding capabilities and torsional flexibility are important determinants of its effect on transcription in both enzymes. These influence the kinetics of lesion bypass and may well play a role in TM and TCR in cells.
Department of Biology, New York University, New York, NY 10003-6688, USA.
Alexandra
A
Dimitri
Lei
L
Jia
Vladimir
V
Shafirovich
Nicholas E
NE
Geacintov
Suse
S
Broyde
David A
DA
Scicchitano
D001483:Base Sequence; D004247:DNA / Q000187:drug effects*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006146:Guanidines / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D009593:Nitroimidazoles / Q000494:pharmacology*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
C493908:5-guanidino-4-nitroimidazole; D006146:Guanidines; D009593:Nitroimidazoles; D012333:RNA, Messenger; D014764:Viral Proteins; D004247:DNA; D012319:RNA Polymerase II; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases