DNA topoisomerase III from extremely thermophilic archaebacteria. ATP-independent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature.

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase. It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA. At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA. At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair.

The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated lambda in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5. While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.

[Action of plasmid ColIb-P9 on the survival after ultraviolet irradiation and on the mutagenesis of the imiC, uvm, recL, uvrE and tif1 sfiA lexA spr mutants of Escherichia coli K-12 cells]. / Deistvie plazmidy ColIb-P9 na vyzhivaemost' posle ul'trafioletovogo oblucheniia i mutagenez imiC, uvm, recL, uvrE, tif1 sfiA LexA spr-mutantov kletok Escherichia coli K-12.

To clarify the mechanisms whereby the ColIb-P9 plasmid affects DNA repair processes, its effect was studied in mutant Escherichia coli K-12 cells with altered mutagenesis and DNA repair. The plasmid was shown to protect umuC, uvm, recL and uvrE mutants after UV irradiation. The frequency of UV-induced his+ revertants increased in the presence of the plasmid in umuC, uvm and recL mutant cells. The ColIb-P9 plasmid completely restored the UV mutability and survival of umuC mutants. These results suggest that the ColIb-P9 plasmid may encode a product similar to that of the umuC gene. In the tif1 sfiA lexA spr mutant cells where SOS functions are constitutively expressed, the ColIb-P9 plasmid increased the number of his+ revertants several times. This suggests that the action of ColIb-P9 is probably brought about not via the derepression of the recA gene but at the subsequent stages of the recA+lexA+-dependent DNA error-prone repair.

The SOS system of Escherichia coli in the regulation of bacteriophage lambda development.

A sequence homologous to the known SOS boxes is found in the Po promoter of phage lambda. It is suggested that the sequence found is a binding site for the LexA repressor. The mechanism of the LexA part in regulation of lambda development is discussed. It is based on the competitive transcription of the RNA encoding CII protein and the short OOP-RNA transcribed from the Po promoter.

[Reactivation of ultraviolet-irradiated phage lambda and recombination in Escherichia coli K-12 cells containing plasmid ColIb-P9]. / Reaktivatsiia obluchennogo ul'trafioletom faga lambda i rekombinatsiia v kletkakh Escherichia coli K-12, soderzhashchikh plazmidy ColIb-P9.

It was shown that the presence of colicinogenis plasmid ColIb-P9 increased the survival of UV-irradiated bacteriophage lambda cI857 in non-irradiated cells of Escherichia coli K-12. The effect of this plasmid was retained in the polA and recB mutants, being sharply reduced in the uvrA and recB recC sbcB recF mutants. This effect strongly depended on recA+ and lexA+ genotype. The W-reactivation efficiency was slightly higher in the cells containing ColIb-P9 than in those lacking the plasmid. No significant effect of the plasmid on recombination during transduction, after conjugation under usual conditions and in the case when a conjugation mixture or recipient cells were irradiated, was observed. The data demonstrate that the effect of ColIb-P9 plasmid on DNA repair is not mediated by its influence on recombination.

The influence of colicinogenic plasmids ColIb-P9, ColIa-CA53 and ColV-K30 on the repair, mutagenesis and induction of colicin E1 synthesis.

The presence of colicinogenic plasmids ColIb-P9 and ColIa-CA53 in E. coli K-12 cells, wild-type with respect to repair, enhanced the survival of cells after UV irradiation and increased the frequency of UV-induced argE3 and his-4 reversions, while the presence of ColV-K30 negatively affected repair and mutagenesis. The plasmid ColIb-P9 showed a UV-protective effect in E. coli cells carrying mutations in genes uvrA, uvrB, uvrC, polA, recB, recF, though in none of the mutants did cell survival reach the wild-type level. The effect of ColIb-P9 on mutagenesis did not depend on the uvrA or recB genes. The plasmid's protective effect and the enhancement of mutagenesis depended on the recA+ lexA+ genotype. The frequency of 2-aminopurine-induced mutations was not affected by ColIb-P9 or ColV-K30. The presence of ColIb-P9 decreased the ability of ColE1-carrying cells to induce colicin E1 synthesis caused by DNA-damaging agents: UV, MNNG, mitomycin C, whereas ColV-K30 increased the percentage of colicin E1-producing cells. These plasmid effects on the level of induction of colicin E1 synthesis were not observed in the case of induction caused by chloramphenicol which did not depend on the products of recA and lexA genes.

[Effect of colicinogenic plasmids on mutagenesis and the induction of colicin E1 synthesis]. / Vliianie kolitsinogennykh plazmid na mutagenez i induktsiiu sinteza kolitsina E1.

The frequency of UV-induced arg+ and his+ reversions is found to be enhanced by the presence of colicinogenic plasmids Ib-P9 and Ia-CA53 and reduced by the presence of plasmid V-K30. The effect of Ib-P9 on the UV-induced mutagenesis depended on recA and lexA genes and was independent on uvrA and recB genes. Plasmids Ib-P9 and V-K30 proved to exert no influence on the frequency of 2-aminopurine-induced mutations. The presence of Ib-P9 reduced the ability of cells carrying the colicinogenic factor E1 to induce colicin E1 synthesis under the action of DNA-damaged agents (UV, nitrosoguanidine, mitomycin C), while plasmid V-K30 enhanced the induction of colicin E1 synthesis. However this effect of plasmids on the induction of colicin E1 synthesis was not observed when the induction occurred under the action of chloramphenicol. Possible mechanisms governing the participation of plasmid gene products in processes related to the repair of DNA are discussed.

[Protective action of colicinogenic factor Ib-P9 on Escherichia coli cells defective in known repair functions after ultraviolet irradiation]. / Zashchitnoe deistvie kolitsinogennogo faktora Ib-P9 na kletki Escherichia coli, defektnye po izvestnym reparativnym funktsiiam, posle ul'trafioletovogo oblucheniia.

The presence of the plasmid colicinogenic factor Ib-P9 in Escherichia coli wild type cells is shown to increase bacterial survival after UV irradiation and the action of N-methyl-N'-nitro-N-nitrosoguanidine. The ability of the plasmid to cause the UV protection is observed in uvrA, uvrB, uvrC, polA, recB, recF E. coli strains, but the plasmid does not restore the UV resistance of the mutant cells to the wild type level. The protective effect of the plasmid CoI Ib-P9 depends on the recA+lexA+ genotype of the cells. The inhibition of protein synthesis (amino acid starvation) before and after UV irradiation does not prevent the UV protection by ColIb-P9. The nature of the plasmid-associated repair functions is discussed.