8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G → T and A → C substitutions

Keith Cheng, D. S. Cahill, H. Kasai, S. Nishimura, L. A. Loeb

Research output: Contribution to journalArticle

1576 Citations (Scopus)

Abstract

Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G → T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A → C substitutions, and two were G → T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G → T substitutions and misincorporation of oh8Gua as substrate causing A → C substitutions, both caused by oh8Gua·A mispairs.

Original languageEnglish (US)
Pages (from-to)166-172
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number1
StatePublished - Jan 1 1992

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DNA Damage
Substitution reactions
Assays
Mutation
DNA
Viral Plaque Assay
Nucleotides
Single-Stranded DNA
Guanine
Mutation Rate
DNA-Directed DNA Polymerase
Bacteriophages
Base Pairing
Polymerization
Substrates
Research Design
Design of experiments
Color
8-hydroxyguanine

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cheng, Keith ; Cahill, D. S. ; Kasai, H. ; Nishimura, S. ; Loeb, L. A. / 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G → T and A → C substitutions. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 1. pp. 166-172.
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8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G → T and A → C substitutions. / Cheng, Keith; Cahill, D. S.; Kasai, H.; Nishimura, S.; Loeb, L. A.

In: Journal of Biological Chemistry, Vol. 267, No. 1, 01.01.1992, p. 166-172.

Research output: Contribution to journalArticle

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AB - Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G → T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A → C substitutions, and two were G → T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G → T substitutions and misincorporation of oh8Gua as substrate causing A → C substitutions, both caused by oh8Gua·A mispairs.

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