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Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases.

Abstract
Chronic inflammation involving constant generation of nitric oxide (*NO) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2'-deoxyinosine (dI) and 2'-deoxyxanthosine are primarily formed by *NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol alpha was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol eta and pol kappaDeltaC (a truncated form of pol kappa) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol alpha, pol eta, and pol kappaDeltaC, promoted preferential incorporation of 2'-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2'-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol alpha, pol eta, and pol kappaDeltaC indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A-->G transition. This ()NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis.
AuthorsManabu Yasui, Emi Suenaga, Naoki Koyama, Chikahide Masutani, Fumio Hanaoka, Petr Gruz, Shinya Shibutani, Takehiko Nohmi, Makoto Hayashi, Masamitsu Honma
JournalJournal of molecular biology (J Mol Biol) Vol. 377 Issue 4 Pg. 1015-23 (Apr 04 2008) ISSN: 1089-8638 [Electronic] Netherlands
PMID18304575 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • DNA Adducts
  • DNA Primers
  • Nitric Oxide
  • Inosine
  • DNA
  • DNA-Directed DNA Polymerase
  • deoxyinosine
Topics
  • Base Sequence
  • DNA (biosynthesis)
  • DNA Adducts (metabolism, pharmacology)
  • DNA Primers (metabolism)
  • DNA Repair (drug effects)
  • DNA Replication (drug effects, physiology)
  • DNA-Directed DNA Polymerase (metabolism, physiology)
  • Humans
  • Inosine (analogs & derivatives, genetics, pharmacology)
  • Models, Biological
  • Molecular Sequence Data
  • Mutation (physiology)
  • Nitric Oxide (metabolism, pharmacology)
  • Substrate Specificity
  • Templates, Genetic

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