Dual role of NER in mutagenesis in Pseudomonas putida.

Nucleotide excision repair (NER) is one of the most important repair systems which counteracts different forms of DNA damage either induced by various chemicals or irradiation. At the same time, less is known about the functions of NER in repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in Pseudomonas putida. The genome of this organism contains two uvrA genes, uvrA and uvrA2. Genetic studies on the effects of uvrA, uvrA2, uvrB and UvrC in mutagenic processes revealed that all of these genes are responsible for the repair of UV-induced DNA damage in P. putida. However, uvrA plays more important role in this process than uvrA2 since the deletion of uvrA2 gene had an effect on the UV-tolerance of bacteria only in the case when uvrA was also inactivated. Interestingly, the lack of functional uvrB, uvrC or uvrA2 gene reduced the frequency of stationary-phase mutations. The contribution of uvrA2, uvrB and uvrC to the mutagenesis appeared to be most significant in the case of 1-bp deletions whose emergence is dependent on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida-besides functioning in repair of damaged DNA, NER is also important in generation of mutations. We hypothesize that NER enzymes may initiate gratuitous DNA repair and the following DNA repair synthesis might be mutagenic.

Study of involvement of ImuB and DnaE2 in stationary-phase mutagenesis in Pseudomonas putida.

Several bacterial species carry in their genomes a so-called "mutagenesis" gene cluster encoding ImuB which is similar to Y-family DNA polymerases, and DnaE2 related to the catalytic subunit DnaE of Pol III. Y-family DNA polymerases are known to be involved in stationary-phase mutagenesis and DnaE2 homologues characterized so far have expressed a mutator phenotype. In this study, we raised a question about the involvement of ImuB and DnaE2 in stationary-phase mutagenesis. Here, we show that Pseudomonas putida ImuB and DnaE2 have antagonistic effects on stationary-phase mutagenesis. ImuB facilitated accumulation of stationary-phase mutants up to two-fold. In contrast to that, DnaE2 had no significant effect on emergence of 1-bp deletion mutants and moreover, it acted as an anti-mutator in accumulation of base substitution mutants in starving bacteria. Similar antagonistic effects of DnaE2 and ImuB on mutagenesis appeared also in UV-mutagenesis study. This data distinguishes the DnaE2 of P. putida from its homologues studied in other organisms.