Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA

Vladimir Shafirovich,Konstantin Kropachev,Thomas Anderson,Zhi Liu,Marina Kolbanovskiy,Brooke D Martin,Kent Sugden,Yoonjung Shim,Xuejing Chen,Jung-Hyun Min,Nicholas E Geacintov

doi:10.1074/jbc.m115.693218

Abstract

The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC(-/-) fibroblast cell extracts. However, normal NER activity appeared when the XPC(-/-) cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1(-/-) relative to NEIL1(+/+) extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions.

Highlights

Nine is 8-oxo-7,8-dihydroguanine (8-oxoG),3 which is ubiquitous in cellular DNA and is used widely as a biomarker of oxidative stress [7]
We demonstrate that the incubation of site- modified oligonucleotide duplexes containing single Sp or Gh lesions are excised by the human nucleotide excision repair (NER) and base excision repair (BER) systems when incubated in cell-free HeLa S3 cell extracts, whereas NIm is resistant to NER but is a substrate of BER
The results shown in this gel were obtained from experiments that were conducted with aliquots from the same cell extract and at the same time; it is, possible to evaluate the relative incision activities of different substrates from such gels

Summary

Introduction

Nine is 8-oxo-7,8-dihydroguanine (8-oxoG),3 which is ubiquitous in cellular DNA and is used widely as a biomarker of oxidative stress [7]. We demonstrate that the incubation of site- modified oligonucleotide duplexes containing single Sp or Gh lesions are excised by the human NER and BER systems when incubated in cell-free HeLa S3 cell extracts, whereas NIm is resistant to NER but is a substrate of BER.

Results

Conclusion