Kazuo Negishi

HAM1, the gene controlling 6-N-hydroxylaminopurine sensitivity and mutagenesis in the yeast Saccharomyces cerevisiae

The ham1 mutant of yeast Saccharomyces cerevisiae is sensitive to the mutagenic and lethal effects of the base analog, 6‐N‐hydroxylaminopurine (HAP). We have isolated a clone from a centromere‐plasmid‐based genomic library complementing HAP sensitivity of the ham1 strain. After subcloning, a 3·4 kb functional fragment was sequenced. It contained three open reading frames (ORFs) corresponding to proteins 353, 197 and 184 amino acids long. LEU2+ disruptions of the promoter and N‐terminal part of the gene coding 197 amino acids long protein led to moderate and strong sensitivity to HAP, respectively, and were allelic to the original ham1‐1 mutation. Thus this ORF represents the HAM1 gene. The deduced amino acid sequence of HAM1 protein was not similar to any protein sequence of the SwissProt database. The HAM1 gene was localized on the right arm of chromosome X between cdc8 and cdc11. Spontaneous mutagenesis was not affected by the ham1::LEU2 disruption mutation.

Spectra of superoxide-induced mutations in the lacI gene of a wild-type and a mutM strain of Escherichia coli K-12

We have analyzed the spectra of superoxide-induced mutations in the chromosomal lacI gene of a wild-type and a mutM strain of Escherichia coli K-12. The mutM strain is known to be deficient in removing 8-hydroxyguanine from DNA. An intracellular superoxide-generating agent, menadione, was used to cause the mutation. Analysis of the mutated DNA showed marked differences between the mutants from the wild type and those from the mutM strain. In the mutants from the wild type, all possible base-pair substitutions were present and their proportions were similar to each other, whereas in those from the mutM bacteria there was a 90% bias in favor of transversion. Furthermore, in the mutM strain GC-to-CG transversion rather than GC-to-TA was predominantly induced. 64% of the GC-to-CG transversions in the mutM strain occurred at the site of (CT/GC)GGC (mutated base underlined). The favorable mutation site, CTGGC, was the same as that of the UV- and sunlight-induced mutations previously reported: the mutations observed there were also G-to-C transversions. We speculate from these results that the superoxide in the cells may lead to production of a modified guanine that can pair with guanine and is subject to removal by the MutM protein.

GENERATION OF INTRACELLULAR ACTIVE OXYGENS IN MOUSE FM3A CELLS BY 3-HYDROXYAMINO-1-METHYL-5H-PYRIDO[4,3-b]INDOLE, THE ACTIVATED TRP-P-2

Mouse FM3A cells in culture were treated with a reactive metabolite of 3‐amino‐1‐methyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐2), 3‐hydroxyamino‐1‐methyl ‐ 5H ‐ pyrido[4,3 ‐b]indole (Trp ‐ P ‐ 2‐ (NHOH)). When the treated cells, which were judged as viable on the basis of trypan‐blue exclusion, were subjected to nitroblue tetrazolium staining, formazan was formed inside the cells, a fact suggesting the intracellular presence of superoxide. No formazan formation was detected on treatment of the cells with Trp‐P‐2. Single‐strand breaks in the cellular DNA took place during this treatment with Trp‐P‐2(NHOH). Since Trp‐P‐2(NHOH) in solution generates superoxide anion accompanying its oxidative degradation, we conclude that the Trp‐P‐2(NHOH) treatment produces intracellular active oxygens that can damage DNA.

Nucleoside and nucleobase analog mutagens

Compounds with structures close to those of normal nucleosides or nucleobases may be incorporated into cells and then become constituents of their DNA. Proliferation of such cells could yield mutants. In this article, the current status of studies on such nucleoside and nucleobase analogs is described. Base mispairing mechanisms for these analogs are discussed in light of recent biochemical and biophysical findings.

Mutagenicity of 5-bromouracil and N6-hydroxyadenine studied by yeast oligonucleotide transformation assay

The mutagenicity of 5-bromouracil (BrU) and N6-hydroxyadenine (HA) was tested by means of the yeast oligonucleotide transformation procedure. BrU-containing oligonucleotide was not mutagenic; although two mutants (per 200 micrograms oligonucleotide) were obtained, they were attributed to base insertion or base substitution at positions different from BrU. This result supports the view that BrU mutagenesis is dependent on intracellular nucleotide pool imbalance. In contrast, HA-containing oligonucleotide was highly mutagenic; 56 mutants (per 140 micrograms oligonucleotide) were obtained. Of 21 induced mutants examined, 20 had G and one had C at the HA position, a result indicating that HA-->G changes took place. To provide back-up evidence, we carried out a general reversion assay for base HA using a set of yeast tester strains, and the results showed that HA induces exclusively AT-to-GC and GC-to-AT transitions. We conclude that in S. cerevisiae HA is a classic base analog mutagen, causing AT-to-GC and GC-to-AT transitions by ambiguous base pairing. The present work has clearly demonstrated the usefulness of the oligonucleotide transformation procedure for elucidating mutagenicity of modified bases.

Template properties of mutagenic cytosine analogues in reverse transcription

We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome.

A new reaction useful for chemical cross-linking between nucleic acids and proteins

Cytosine in nucleic acids can be converted into N 4‐aminocytosine by treatment with a mixture of hydrazine and bisulfite. The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker. Successful use of this scheme of chemical cross‐linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E. coli 30 S ribosomal subunit.

Highly efficient random mutagenesis in transcription-reverse-transcription cycles by a hydrogen bond ambivalent nucleoside 5'-triphosphate analogue: potential candidates for a selective anti-retroviral therapy.

The nucleoside P can base pair with both A and G. We evaluated the mutation frequency induced by the 5′-triphospbate of the ribonucleoside P (PTP) in an in vitro retroviral replication model. After 4 cycles of replication in the presence of PTP, the mutation frequency was raised to 3.8 × 10−2 per nucleotide and C-to-U and U-to-C mutations were dominantly observed. These results suggest that ambivalent NTP analogues, like PTP, could induce mutations beyond the error threshold of retroviruses.

Inhibition of DNA Adduct Formation and Mutagenic Action of 3-Amino-1-methyl-5H-pyrido[4,3-b]indole by Chlorophyllin-chitosan in rpsL Transgenic Mice

We have studied the inhibitory effect of chlorophyllin‐chitosan (Chl‐Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen‐carcinogen, 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐2), in mice carrying the E. coli rpsL gene as a mutagenesis reporter. Upon administration of a diet containing 0.002% or 0.01% Trp‐P‐2, DNA adducts were formed in various tissues in a dose‐dependent manner, with the maximum level observed in the liver. Addition of 3% Chl‐Chi to the diet reduced the Trp‐P‐2 adduct by up to 90%. The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp‐P‐2 diet. Addition of Chl‐Chi to the diet decreased these induced mutant frequencies to the background level. No harmful effect of Chl‐Chi was detected during these experiments. The results show that Chl‐Chi may be a candidate chemopreventive agent against the genotoxic action of Trp‐P‐2, and possibly also other aromatic carcinogens in the diet.

Deoxyribonucleoside 3′-phosphorobisamidites in the synthesis of isopropyl phosphotriester oligodeoxyribonucleotide analogues

Abstract Deoxyribonucleoside phosphorobisdiethylamidites were prepareded by using bis(diethylamino)phosphorochloridite as a phosphitylating reagent. These intermediates were used for the solid-phase synthesis of oligodeoxyribonucleotides with isopropyl phosphotriester at the designated position. The diastereoisomers of the modified oligomers were separated by reversed-phase HPLC and their absolute configulations were determined by chemical correlation with the known Rp ans Sp phosphothioate analogue of oligodeoxyribonucleotide.