Differential blocking effects of the acetaldehyde-derived DNA lesion N2-ethyl-2'-deoxyguanosine on transcription by multisubunit and single subunit RNA polymerases.
Acetaldehyde, the first metabolite of ethanol, reacts with DNA to form adducts, including N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG). Although the effects of N(2)-Et-dG on DNA polymerases have been well studied, nothing is known about possible effects of this lesion on transcription by RNA polymerases (RNAPs). Using primer extension assays in vitro, we found that a single N(2)-Et-dG lesion is a strong block to both mammalian RNAPII and two other multisubunit RNAPs, (yeast RNAPII and Escherichia coli RNAP), as well as to T7 RNAP. However, the mechanism of transcription blockage appears to differ between the multisubunit RNAPs and T7 RNAP. Specifically, all three of the multisubunit RNAPs can incorporate a single rNTP residue opposite the lesion, whereas T7 RNAP is essentially unable to do so. Using the mammalian RNAPII, we found that CMP is exclusively incorporated opposite the N(2)-Et-dG lesion. In addition, we also show that the accessory transcription factor TFIIS does not act as a lesion bypass factor, as it does for other nonbulky DNA lesions; instead, it stimulates the polymerase to remove the CMP incorporated opposite the lesion by mammalian RNAPII. We also include models of the N(2)-Et-dG within the active site of yeast RNAPII, which are compatible with our observations.
Cheng
Tsu-Fan
TF
Section on Molecular Neurobiology, Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892.
Hu
Xiaopeng
X
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201.
Gnatt
Averell
A
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201.
Brooks
Philip J
PJ
Section on Molecular Neurobiology, Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892. Electronic address: pjbrooks@mail.nih.gov.
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D014158:Transcription, Genetic / Q000502:physiology*; D035602:Transcriptional Elongation Factors / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry*
D029968:Escherichia coli Proteins; C492934:N2-ethyl-2'-deoxyguanosine; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; D014764:Viral Proteins; C018648:transcription factor S-II; D004247:DNA; D012319:RNA Polymerase II; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003849:Deoxyguanosine