Biochemistry. 2017 Jun 20;56(24):3008-3018. doi: 10.1021/acs.biochem.7b00295. 

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

Kolbanovskiy M1, Chowdhury MA2, Nadkarni A2, Broyde S2, Geacintov NE1, Scicchitano DA2,3, Shafirovich V1.

1 Department of Chemistry, New York University , 100 Washington Square East, New York, New York 10003-5180, United States.
2 Department of Biology, New York University , 100 Washington Square East, New York, New York 10003-5180, United States.
3 Division of Science, New York University Abu Dhabi , P.O. Box 129188, Abu Dhabi, United Arab Emirates.

 

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.
PMID: 28514164