Silvia Ayora

Bacillus subtilis homologous recombination: genes and products

Homologous recombination plays a critical role in maintaining gene diversification and genome stability. Fourteen Bacillus subtilis recombination gene products have been genetically characterised and classified into five different epistatic groups. At least seven other recombination genes could be predicted. Recombination gene products which define activities that help RecA to process DNA repair and recombination have been studied, but those that processed recombination intermediates into products (post-synaptic stage) await elucidation.

Genetic Recombination in Bacillus subtilis 168: Contribution of Holliday Junction Processing Functions in Chromosome Segregation

Bacillus subtilis mutants classified within the epsilon (ruvA, DeltaruvB, DeltarecU, and recD) and eta (DeltarecG) epistatic groups, in an otherwise rec+ background, render cells impaired in chromosomal segregation. A less-pronounced segregation defect in DeltarecA and Deltasms (DeltaradA) cells was observed. The repair deficiency of addAB, DeltarecO, DeltarecR, recH, DeltarecS, and DeltasubA cells did not correlate with a chromosomal segregation defect. The sensitivity of epsilon epistatic group mutants to DNA-damaging agents correlates with ongoing DNA replication at the time of exposure to the agents. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect in ruvA and recD cells and the segregation defect in ruvA and DeltarecG cells. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect of DeltarecU cells but do not suppress the segregation defect in these cells. The DeltarecA mutation suppresses the segregation defect but does not suppress the DNA repair defect in DeltarecU cells. These results result suggest that (i) the RuvAB and RecG branch migrating DNA helicases, the RecU Holliday junction (HJ) resolvase, and RecD bias HJ resolution towards noncrossovers and that (ii) Sms (RadA) and SubA proteins might play a role in the stabilization and or processing of HJ intermediates.

Bacillus subtilis RecU Holliday-junction resolvase modulates RecA activities

The Bacillus subtilis RecU protein is able to catalyze in vitro DNA strand annealing and Holliday-junction resolution. The interaction between the RecA and RecU proteins, in the presence or absence of a single-stranded binding (SSB) protein, was studied. Substoichiometric amounts of RecU enhanced RecA loading onto single-stranded DNA (ssDNA) and stimulated RecA-catalyzed D-loop formation. However, RecU inhibited the RecA-mediated three-strand exchange reaction and ssDNA-dependent dATP or rATP hydrolysis. The addition of an SSB protein did not reverse the negative effect exerted by RecU on RecA function. Annealing of circular ssDNA and homologous linear 3′-tailed double-stranded DNA by RecU was not affected by the addition of RecA both in the presence and in the absence of SSB. We propose that RecU modulates RecA activities by promoting RecA-catalyzed strand invasion and inhibiting RecA-mediated branch migration, by preventing RecA filament disassembly, and suggest a potential mechanism for the control of resolvasome assembly.

Evidence for Different Pathways during Horizontal Gene Transfer in Competent Bacillus subtilis Cells

Cytological and genetic evidence suggests that the Bacillus subtilis DNA uptake machinery localizes at a single cell pole and takes up single-stranded (ss) DNA. The integration of homologous donor DNA into the recipient chromosome requires RecA, while plasmid establishment, which is independent of RecA, requires at least RecO and RecU. RecA and RecN colocalize at the polar DNA uptake machinery, from which RecA forms filamentous structures, termed threads, in the presence of chromosomal DNA. We show that the transformation of chromosomal and of plasmid DNA follows distinct pathways. In the absence of DNA, RecU accumulated at a single cell pole in competent cells, dependent on RecA. Upon addition of any kind of DNA, RecA formed highly dynamic thread structures, which rapidly grew and shrank, and RecU dissipated from the pole. RecO visibly accumulated at the cell pole only upon addition of plasmid DNA, and, to a lesser degree, of phage DNA, but not of chromosomal DNA. RecO accumulation was weakly influenced by RecN, but not by RecA. RecO annealed ssDNA complexed with SsbA in vitro, independent of any nucleotide cofactor. The DNA end-joining Ku protein was also found to play a role in viral and plasmid transformation. On the other hand, transfection with SPP1 phage DNA required functions from both chromosomal and plasmid transformation pathways. The findings show that competent bacterial cells possess a dynamic DNA recombination machinery that responds in a differential manner depending if entering DNA shows homology with recipient DNA or has self-annealing potential. Transformation with chromosomal DNA only requires RecA, which forms dynamic filamentous structures that may mediate homology search and DNA strand invasion. Establishment of circular plasmid DNA requires accumulation of RecO at the competence pole, most likely mediating single-strand annealing, and RecU, which possibly down-regulates RecA. Transfection with SPP1 viral DNA follows an intermediate route that contains functions from both chromosomal and plasmid transformation pathways.

Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Subsaturating amounts of Bacillus subtilis SsbA, independently of the order of addition, partially inhibit the single-stranded DNA-dependent dATPase activity of RecA. This negative effect is fully overcome when a substoichiometric amount of RecO is added. SsbA added prior to RecA does not stimulate the dATP-dependent DNA strand exchange activity; however, added after RecA it enhances the extent of strand exchange. The addition of RecO stimulates RecA-mediated joint molecule formation, although it limits the accumulation of final recombination products. Thus we suggest that RecO has a dual activity: RecO acts as a RecA mediator enabling RecA to utilize SsbA-coated single-stranded DNA as a polymerization substrate and controls RecA-mediated DNA strand exchange by limiting its extent. We herein discuss the possible mechanisms of RecO involvement in the regulation of double strand break repair and genetic transformation.

Recombination-dependent concatemeric viral DNA replication

The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.

Plasmid pSM19035, a model to study stable maintenance in Firmicutes.

pSM19035 is a low-copy-number theta-replicating plasmid, which belongs to the Inc18 family. Plasmids of this family, which show a modular organization, are functional in evolutionarily diverse bacterial species of the Firmicutes Phylum. This review summarizes our understanding, accumulated during the last 20 years, on the genetics, biochemistry, cytology and physiology of the five pSM19035 segregation (seg) loci, which map outside of the minimal replicon. The segA locus plays a role both in maximizing plasmid random segregation, and in avoiding replication fork collapses in those plasmids with long inverted repeated regions. The segB1 locus, which acts as the ultimate determinant of plasmid maintenance, encodes a short-lived epsilon(2) antitoxin protein and a long-lived zeta toxin protein, which form a complex that neutralizes zeta toxicity. The cells that do not receive a copy of the plasmid halt their proliferation upon decay of the epsilon(2) antitoxin. The segB2 locus, which encodes two trans-acting, ParA- and ParB-like proteins and six cis-acting parS centromeres, actively ensures equal or roughly equal distribution of plasmid copies to daughter cells. The segC locus includes functions that promote the shift from the use of DNA polymerase I to the replicase (PolC-PolE DNA polymerases). The segD locus, which encodes a trans-acting transcriptional repressor, omega(2), and six cis-acting cognate sites, coordinates the expression of genes that control copy number, better-than-random segregation and partition, and assures the proper balance of these different functions. Working in concert the five different loci achieve almost absolute plasmid maintenance with a minimal growth penalty.

Bacillus subtilis SsbA and dATP regulate RecA nucleation onto single-stranded DNA.

Bacillus subtilis RecA preferentially hydrolyzes dATP over ATP and supports an efficient DNA strand exchange reaction in the presence of dATP when compared to ATP. Saturating amounts of SsbA, independently of the order of addition, reduce the single-stranded (ss) DNA-dependent dATPase activity of RecA, and block the ATPase activity. SsbA added prior to RecA slightly stimulates the dATP-dependent DNA strand exchange activity, whereas added after RecA greatly enhances the extent of strand exchange. In the presence of ATP, 10 times more RecA is required to achieve a comparable level of strand exchange than in the presence of dATP. We propose that dATP binding and hydrolysis as well as SsbA provide different levels of regulation of the dynamic RecA nucleoprotein filament.

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protects ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The RecU Holliday junction resolvase acts at early stages of homologous recombination

Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A and RecUR71A do not exert a negative effect on the RecA dATPase and fail to interact with it. Both activities are important in vivo since RecU mutants impaired only in RecA interaction are as sensitive to DNA damaging agents as a deletion mutant.