Miscoding properties of 8-chloro-2'-deoxyguanosine, a hypochlorous acid-induced DNA adduct, catalysed by human DNA polymerases.

Abstract

Many chronic inflammatory conditions are associated with an increased risk of cancer development. At the site of inflammation, cellular DNA is damaged by hypochlorous acid (HOCl), a potent oxidant generated by myeloperoxidase. 8-Chloro-2'-deoxyguanosine (8-Cl-dG) is a major DNA adduct formed by HOCl and has been detected from the liver DNA and urine of rats administered lipopolysaccharide in an inflammation model. Thus, the 8-Cl-dG lesion may be associated with the carcinogenesis of inflamed tissues. In this study, we explored the miscoding properties of the 8-Cl-dG adduct generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotide containing a single 8-Cl-dG was prepared and used as a template in primer extension reactions catalysed by human pol α, ĸ or η. Primer extension reactions catalysed by pol α and ĸ in the presence of all four dNTPs were slightly retarded at the 8-Cl-dG site, while pol η readily bypassed the lesion. The fully extended products were analysed to quantify the miscoding frequency and specificity of 8-Cl-dG using two-phased polyacrylamide gel electrophoresis (PAGE). During the primer extension reaction in the presence of four dNTPs, pol ĸ promoted one-base deletion (6.4%), accompanied by the misincorporation of 2'-deoxyguanosine monophosphate (5.5%), dAMP (3.7%), and dTMP (3.5%) opposite the lesion. Pol α and η, on the other hand, exclusively incorporated dCMP opposite the lesion. The steady-state kinetic studies supported the results obtained from the two-phased PAGE assay. These results indicate that 8-Cl-dG is a mutagenic lesion; the miscoding frequency and specificity varies depending on the DNA polymerase used. Thus, HOCl-induced 8-Cl-dG adduct may be involved in inflammation-driven carcinogenesis.

• Authors: Akira Sassa, Nagisa Kamoshita, Tomonari Matsuda, Yuji Ishii, Isao Kuraoka, Takehiko Nohmi, Toshihiro Ohta, Masamitsu Honma, Manabu Yasui
• Journal: Mutagenesis (Mutagenesis) Vol. 28 Issue 1 Pg. 81-8 (Jan 2013) ISSN: 1464-3804 [Electronic] England
• PMID: 23076070 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 8-chloro-2'-deoxyguanosine
• DNA Adducts
• Oligodeoxyribonucleotides
• DNA Polymerase I
• DNA-Directed DNA Polymerase
• Deoxyguanosine

Topics

• DNA Adducts (chemistry, metabolism)
• DNA Polymerase I (chemistry, metabolism)
• DNA-Directed DNA Polymerase (chemistry, metabolism)
• Deoxyguanosine (analogs & derivatives, chemistry, metabolism)
• Electrophoresis, Gel, Two-Dimensional
• Humans
• Kinetics
• Oligodeoxyribonucleotides (chemistry)