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Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II.
DNA Repair (Amst). 2004 May 04; 3(5):483-94.DR

Abstract

8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation. Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell. As a result, several repair processes have evolved to remove this lesion from the genome. It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes. A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR. As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII). We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter. We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95%. Similarly, this lesion only transiently blocked transcription by RNAPII. However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG. When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase. Binding of the Saccharomyces cerevisiae MSH2-MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase. These results are discussed in the context of possible models for TCR.

Authors+Show Affiliations

Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA. silviat@stanford.eduNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15084310

Citation

Tornaletti, Silvia, et al. "Effect of 8-oxoguanine On Transcription Elongation By T7 RNA Polymerase and Mammalian RNA Polymerase II." DNA Repair, vol. 3, no. 5, 2004, pp. 483-94.
Tornaletti S, Maeda LS, Kolodner RD, et al. Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. DNA Repair (Amst). 2004;3(5):483-94.
Tornaletti, S., Maeda, L. S., Kolodner, R. D., & Hanawalt, P. C. (2004). Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. DNA Repair, 3(5), 483-94.
Tornaletti S, et al. Effect of 8-oxoguanine On Transcription Elongation By T7 RNA Polymerase and Mammalian RNA Polymerase II. DNA Repair (Amst). 2004 May 4;3(5):483-94. PubMed PMID: 15084310.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. AU - Tornaletti,Silvia, AU - Maeda,Lauren S, AU - Kolodner,Richard D, AU - Hanawalt,Philip C, PY - 2004/01/20/accepted PY - 2004/4/16/pubmed PY - 2004/12/16/medline PY - 2004/4/16/entrez SP - 483 EP - 94 JF - DNA repair JO - DNA Repair (Amst.) VL - 3 IS - 5 N2 - 8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation. Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell. As a result, several repair processes have evolved to remove this lesion from the genome. It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes. A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR. As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII). We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter. We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95%. Similarly, this lesion only transiently blocked transcription by RNAPII. However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG. When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase. Binding of the Saccharomyces cerevisiae MSH2-MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase. These results are discussed in the context of possible models for TCR. SN - 1568-7864 UR - https://www.unboundmedicine.com/medline/citation/15084310/Effect_of_8_oxoguanine_on_transcription_elongation_by_T7_RNA_polymerase_and_mammalian_RNA_polymerase_II_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1568786404000205 DB - PRIME DP - Unbound Medicine ER -