Celina Janion

The efficiency and extent of mutagenic activity of some new mutagens of base-analogue type

N4-Hydroxycytidine, 5-methyl-N4-hydroxydeoxycytidine and 2-amino-N6-hydroxyadenine were tested for their mutagenic activity in S. typhimurium and E. coli cells. Reversion analysis of different markers was applied in a plate-test system, and 2-aminopurine was used as a reference mutagen. (i) 2-Amino-N6-hydroxyadenine was the most potent mutagen. In some cases it gave more than 1000 colonies of revertants per plate. (ii) N6-Hydroxycytidine was the least specific mutagen. Almost all the tested markers were inducible to revert by this analogue. (iii) The mutagenic specificity of 5-methyl-N4-hydroxydeoxycytidine seemed to be opposite to that of 2-aminopurine. This suggests that the former can induce transition of CG to TA. (iv) A comparison of the mutagenic actions of N4-hydroxycytidine and 5-methyl-N4-hydroxy-deoxycytidine showed that deoxyriboside analogues are not necessarily more efficient mutagens than ribonucleosides. (v) No purine or pyrimidine deficiency was needed for mutagenesis to occur for any of the mutagens investigated. (vi) The results on bacteria with different repair abilities suggest that base-analogue mutagenesis (except perhaps for BrdUrd) occurs mainly during replication of nucleic acids containing substituted nucleosides with bi-functional specificity.

Mutagenic and inhibitory properties of some new purine analogs on Salmonella typhimurium TA1530

Mutagenic and growth-inhibitory effects of 2-amino-N6-hydroxyadenine, 2-amino-N6-methoxyadenine and 2-amino-N6-methyl-N6-hydroxyadenine were examined on S. typhimurium TA1530. All compounds showed strong mutagenic activity, and 2-amino-N6-hydroxyadenine induced mutations at a dose as low as 1 microgram/ml. 2-Amino-N6-hydroxyadenine and, to a lesser extent, 2-amino-N6-methyl-N6-hydroxyadenine (but not 2-amino-N6-methoxyadenine) exerted inhibitory effects on bacterial growth. The specificity of mutagenic action of these base analogs, as well as the target for reversion of the his- marker in TA1530, is discussed.

Mutagenic potency of MMS-induced 1meA/3meC lesions in E. coli.

The mutagenic activity of MMS in E. coli depends on the susceptibility of DNA bases to methylation and their repair by cellular defense systems. Among the lesions in methylated DNA is 1meA/3meC, which is recently recognized as being mutagenic. In this report, special attention is focused on the mutagenic properties of 1meA/3meC which, by the activity of AlkB‐dioxygenase, are quickly and efficiently converted to natural A/C bases in the DNA of E. coli alkB+ strains, preventing 1meA/3meC‐induced mutations. We have found that in the absence of AlkB‐mediated repair, MMS treatment results in an increased frequency of four types of base substitutions: GC→CG, GC→TA, AT→CG, and AT→TA, whereas overproduction of PolV in CC101–106 alkB−/pRW134 strains leads to a markedly elevated level of GC→TA, GC→CG, and AT→TA transversions. It has been observed that in the case of AB1157 alkB− strains, the MMS‐induced and 1meA/3meC‐dependent argE3→Arg+ reversion occurs efficiently, whereas lacZ−→ Lac+ reversion in a set of CC101–106 alkB− strains occurs with much lower frequency. We considered several reasons for this discrepancy, namely, the possible variance in the level of the PolV activity, the effect of the PolIV contents that is higher in CC101–106 than in AB1157 strains and the different genetic cell backgrounds in CC101–106 alkB− and AB1157 alkB− strains, respectively. We postulate that the difference in the number of targets undergoing mutation and different reactivity of MMS with ssDNA and dsDNA are responsible for the high (argE3→Arg+) and low (lacZ− → Lac+) frequency of MMS—induced mutations. Environ. Mol. Mutagen. 2009.

N4-hydroxycytidine-a new mutagen of a base analogue type.

Abstract N 4 -hydroxycytidine (N 4 -OHcyd) ∗ is incorporated into nucleic acids of a cytidine-requiring strain of S.typhimurium 1045 and can act mutagenically. The reversion frequency of pyrG − → pyrG + is 10–20 fold higher than the spontaneous background. N 4 OHcyd-induced revertants show a strong inhibitory effect in the presence of N 4 OHcyd. The influence of N 4 OHcyd on cytidine metabolism is discussed.

N4-hydroxycytidine: A mutagen specific for at to GC transitions

N4-Hydroxycytidine is a mutagen of the base-analog type and one of the products formed by treatment of cytidine with hydroxylamine. In this communication evidence is presented showing that, in contrast with other known base analogs, N4-hydroxycytidine results mainly, if not exclusively, in AT leads to GC transitional alterations in Escherichia coli K12.

Alternative pathways of methyl methanesulfonate-induced mutagenesis in Escherichia coli.

SummaryMethyl methanesulfonate (MMS) induced mutagenesis is known to be largely dependent on functional umuCD and recA genes. By phenotypic analysis of Arg+ (argE3, ochre) revertants according to their reversion of the mutations his-4 (ochre) and thr-1 (amber), we attempted to deduce the specificity and/or sites of MMS-induced mutations. It is shown that: (1) MMS-induced, umuC-dependent Arg+ revertants (which prevail in bacteria proficient in mismatch repair) result from a different mutational pathway from umuC-independent ones. UmuC-dependent Arg+ revertants belong to class 2 (Arg+His+Thr−), and umuC-independent ones to class 1 (Arg+His−Thr−). (2) The mismatch repair system very efficiently prevents mutations induced by MMS. We found that in the mutS strain, deficient in mismatch repair, class 1 Arg+ revertants are the most numerous, whereas class 2 Arg+ revertants occur at similar levels in MMS-treated mutS and mutS+ strains. Therefore the mismatch repair system very efficiently prevents formation of umuC-independent Arg+ revertants, but exerts negligible or no effect on umuC-dependent Arg+ revertants. (iii) Both mutS umuC and mutS recA strains, are highly mutable by MMS.

Ability of base analogs to induce the SOS response: effect of a dam mutation and mismatch repair system.

Summary2-Aminopurine, 2-amino-N6-hydroxyadenine, 2-amino-N6-methoxyadenine and 2-amino-N6-methyl-N6-hydroxyadenine (but not N4-hydroxycytidine), strong mutagens of base analog type, may induce the SOS response in E. coli cells. This ability is greatly enhanced in dam3 mutants and abolished in dam3mutS, dam3mutH, and dam3mutL strains, thereby suggesting that the mismatch repair system is involved in the mechanism of induction.

Involvement of the mismatch repair system in base analogue-induced mutagenesis

SummaryThe influence of the mismatch repair system, and the role of mutS, mutR, and mutL genes, in mutagenesis induced by n2ome6Ade, n2oh6Ade and n2Pur have been investigated. From the frequency of reversion of Arg−Thr− and His− markers in AB2497, and its mut− derivatives, it was concluded that mismatches introduced by n2-ome6Ade and n2oh6Ade are better substrates for mismatch repair enzymes than that introduced by n2Pur. All these mut-gene products are more active in removing spontaneous or base analogue-induced mismatches which, when unexcised, lead to transversion of base pairs, than those which lead to transitions. Active engagements of mutL, mutR, or mutS gene products depend on the kind of mutation, the site of mutagenesis, and the inducing agent. Dam− cells are over-mutated by both n2ome6Ade and n2oh6Ade, but are hyper-sensitive to n2oh6Ade only. It is proposed that hyper-sensitivity of dam− cells is due not only to an increase in overlaping gap formation on both strands of DNA, but to a greater lability of the impaired cells.Results are presented which strongly suggest that n2-ome6Ade in mut+ cells and n2oh6Ade in mut− only, can induce GC→TA transversions.

Mutagenic specificity of N4-hydroxycytidine

The mutagenic specificty of (oh)4Cyd was examined with T4rII phage mutants which allow for discrimination between AT yields GC and GC yields AT base transitions. AT yields GC transitions were induced with a frequency 1-2 orders of magnitude higher than GC yields AT transitions. The mechanism of this preferential transition pathway is discussed in the light of base-analogue mutagenesis.

Effect of proofreading anddam-instructed mismatch repair systems on N4-hydroxycytidine-induced mutagenesis

SummaryThe role of the proofreading (3′→5′ exonuclease) function of T4 DNA polymerase and the mismatch repair system ofE. coli on N4-hydroxycytidine (oh4Cyd)1 induced mutagenesis was investigated. oh4Cyd-induced mutation is strongly suppressed when the proofreading activity increases as a result of the presence oftsCB87-antimutator polymerase or elevated temperature (43° C vs 30° C). Mutagenic activity of oh4Cyd, however, is little, if at all, affected by the presence of thetsLB56 mutator allele of T4 DNA polymerase with suppressed proofreading activity. This leads to the conclusion that oh4C nucleotides are not frequently removed by proofreading activity of wild-type T4 DNA polymerase. The number of mutations induced by oh4Cyd increases 3- to 5-fold due to damage of the genesmutS,mutL,uvrE, but notmutR.Dam- cells are more sensitive to, and hypermutable by, oh4Cyd in comparison withdam+ cells. This is compatible with the notion that oh4C residues are recognised and excised by mismatch repair enzymes. The results indicate thath neither the proofreading function of T4 DNA polymerase, nor the mismatch repair enzymes, are responsible for the high specificity of oh4Cyd which causes AT→GC transition.