The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro

Marina Kolbanovskiy, Moinuddin A. Chowdhury, Aditi Nadkarni, Suse Broyde, Nicholas E. Geacintov, David A. Scicchitano, Vladimir Shafirovich

Research output: Contribution to journalArticle

Abstract

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.

Original languageEnglish (US)
Pages (from-to)3008-3018
Number of pages11
JournalBiochemistry
Volume56
Issue number24
DOIs
StatePublished - Jun 20 2017

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RNA Polymerase II
Elongation
DNA Repair
Transcription
DNA
Repair
Cell Extracts
guanidinohydantoin
spiroiminodihydantoin
In Vitro Techniques
Polyacrylamide Gel Electrophoresis
Electrophoresis
RNA
Nucleotides
Cells
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro. / Kolbanovskiy, Marina; Chowdhury, Moinuddin A.; Nadkarni, Aditi; Broyde, Suse; Geacintov, Nicholas E.; Scicchitano, David A.; Shafirovich, Vladimir.

In: Biochemistry, Vol. 56, No. 24, 20.06.2017, p. 3008-3018.

Research output: Contribution to journalArticle

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