Rescue of arrested replication forks by homologous recombination.

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

Replication mutations differentially enhance RecA-dependent and RecA-independent recombination between tandem repeats in Bacillus subtilis.

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper-recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA-dependent and RecA-independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.

sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants.

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.

RuvAB acts at arrested replication forks.

Replication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage.

In vivo relations between pAMbeta1-encoded type I topoisomerase and plasmid replication.

A number of large extrachromosomal elements encode prokaryotic type I topoisomerases of unknown functions. Here, we analysed the topoisomerase Topbeta encoded by the Gram-positive broad-host-range plasmid pAMbeta1. We show that this enzyme possesses the DNA relaxation activity of type I topoisomerases. Interestingly, it is active only on plasmids that use DNA polymerase I to initiate replication, such as pAMbeta1, and depends on the activity of this polymerase. This is the first example, to our knowledge, of prokaryotic type I topoisomerase that is specific for a given type of replicon. During pAMbeta1 replication in Bacillus subtilis cells, Topbeta promotes premature arrest of DNA polymerase I, approximately 190bp downstream of the replication initiation point. We propose that Topbeta acts on the early replication intermediates of pAMbeta1, which contain D-loops formed by DNA polymerase I-mediated strand displacement. The possible role of the resulting DNA Pol I arrest in plasmid replication is discussed.

Replication terminus for DNA polymerase I during initiation of pAM beta 1 replication: role of the plasmid-encoded resolution system.

Replication of plasmid pAM beta 1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested approximately 230 bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.

Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication.

We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.

[Subcloning and study of the GTP-cyclohydrolase gene of Bacillus subtilis]. / Subklonirovanie i issledovanie gena GTP-tsiklogidrolazy Bacillus subtilis.

Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells. The position of the gene within the riboflavine operon was defined. The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity.

Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.

[Recombination of plasmids with inverted repeats in Bacillus subtilis cells]. / Rekombinatsiia plazmid s invertirovannymi povtorami v kletkakh Bacillus subtilis.

The plasmid pNB48 carring a pair of genetically distinct inverted repeats is constructed on the basis of plasmid pSM19035 replicon. The single Campbell-type recombination between inverted repeats of two circular monomers leads to the formation of the so called "inverted" dimers in Bacillus subtilis cells. The gene conversion is observed in the course of this recombination. The mechanism of structural rearrangements of plasmids with inverted repeats that was postulated earlier is confirmed now. Some replicon features may influence the recombination of plasmids in Bacillus subtilis cells. The plasmid pNB48 may be a suitable model for studying the genetic recombination in Bacillus subtilis.

[Plasmid inverted repeats provide for duplication or precise excision of DNA inserts]. / Plazmidnye invertirovannye povtory obespechivaiut duplikatsiiu ili tochnoe vyrezanie vstroennykh v nikh fragmentov DNK.

A plasmid containing inverted repeats is constructed in Bacillus subtilis. Insertion of DNA fragments into the plasmid inverted repeats results either in the precise excision of the insert or in its duplication in the opposite inverted repeat. These rearrangements are due to the presence of inverted repeats only. Two recombination events are possibly responsible for these phenomena. During the first step of the recombination two plasmid monomers form a dimer molecule. During the second step the intramolecular recombination between the direct repeats in the dimer structure leads to the formation of two rearranged plasmid monomers devoid of insertion or containing two DNA inserts. Proposed dimeric intermediate is unstable in B. subtilis. However, it is isolated from Escherichia coli recA, cells. Plasmids containing the inverted repeats can serve as a model to study plasmid recombination in B. subtilis cells.