Effects of a guanine-derived formamidopyrimidine lesion on DNA replication. Translesion DNA synthesis, nucleotide insertion, and extension kinetics

Kenjiro Asagoshi, Hiroaki Terato, Yoshihiko Ohyama, Hiroshi Ide

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3′-5′ exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (fins= Vmax/Km for insertion) and extension (fext = Vmax/Km for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative fins = 1) ≫ dTMP (2.4 × 10-4) ≈ dAMP (8.1 × 10-5) > dGMP (4.5 × 10-7)), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative fext = 1) > mFapyG:T (4.6 × 10-3) ≫ mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.

Original languageEnglish
Pages (from-to)14589-14597
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number17
DOIs
Publication statusPublished - Apr 26 2002
Externally publishedYes

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Guanine
DNA Replication
DNA Polymerase I
Nucleotides
Kinetics
DNA
Exonucleases
Oligonucleotides
Reactive Oxygen Species
Escherichia coli
7,8-dihydro-8-oxoguanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of a guanine-derived formamidopyrimidine lesion on DNA replication. Translesion DNA synthesis, nucleotide insertion, and extension kinetics. / Asagoshi, Kenjiro; Terato, Hiroaki; Ohyama, Yoshihiko; Ide, Hiroshi.

In: Journal of Biological Chemistry, Vol. 277, No. 17, 26.04.2002, p. 14589-14597.

Research output: Contribution to journalArticle

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abstract = "2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3′-5′ exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (fins= Vmax/Km for insertion) and extension (fext = Vmax/Km for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative fins = 1) ≫ dTMP (2.4 × 10-4) ≈ dAMP (8.1 × 10-5) > dGMP (4.5 × 10-7)), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative fext = 1) > mFapyG:T (4.6 × 10-3) ≫ mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.",
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