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

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 a 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.

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The vinyl chloride DNA derivative N2,3-ethenoguanine produces G----A transitions in Escherichia coli.

Vinyl chloride is a known human and rodent carcinogen that forms several cyclic base derivatives in DNA. The mutagenic potential of these derivatives has been examined in vitro but not in vivo. One of these derivatives, N2,3-ethenoguanine (epsilon G), is known to base pair with both cytosine and thymine during in vitro DNA synthesis, which would result in G----A transitions. To determine the base pairing specificity of this labile guanine derivative in Escherichia coli, we have developed a genetic reversion assay for guanine derivatives. The assay utilizes DNA polymerase-mediated analogue insertion into a bacteriophage vector, M13G*1, which detects all single-base substitutions at position 141 of the lacZ alpha gene by change in plaque color. After the insertion of a single epsilon G opposite the template cytosine at position 141 by use of epsilon dGTP and DNA polymerase and further extension with all four normal dNTPs, the DNA was transfected into E. coli. Transfection of M13G*1 containing epsilon G at the target site yielded 135 mutants among 26,500 plaques, 134 of which represented G----A transitions. The uncorrected mutation frequency was 0.5%, as compared with the control value, approximately 0.02%; when corrected for epsilon G content and penetrance, the calculated mutagenic potential of epsilon G (mutations/analogue) was about 13%. We thus conclude that epsilon G specifically induces G----A transitions during DNA replication in E. coli. The M13G*1 assay may permit the testing of other labile guanine derivatives not otherwise amenable to mutagenesis studies.