Begoña Carrasco

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 polynucleotide phosphorylase 3′-to-5′ DNase activity is involved in DNA repair

In the presence of Mn2+, an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3′ → 5′ polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn2+ and low-level inorganic phosphate (Pi), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg2+ or high-level Pi. In contrast, the RNase activity of PNPase requires Mg2+ and Pi, suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (ΔpnpA) is not epistatic with ΔrecA, but is epistatic with ΔrecN and Δku, which by themselves are non-epistatic. The addA5, ΔrecO, ΔrecQ (ΔrecJ), ΔrecU and ΔrecG mutations (representative of different epistatic groups), in the context of ΔpnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.

Genetic recombination in Bacillus subtilis: a division of labor between two single-strand DNA-binding proteins

We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.

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.

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.

Bacillus subtilis DprA Recruits RecA onto Single-stranded DNA and Mediates Annealing of Complementary Strands Coated by SsbB and SsbA

Background: Different mediators assist RecA to catalyze genetic recombination. Results: DprA facilitates the displacement of both SSBs (SsbB and SsbA), increases RecA nucleation onto SSB-coated ssDNA, and mediates DNA strand annealing. Conclusion: DprA facilitates RecA-mediated strand exchange and anneals complementary strands coated by an SSB protein. Significance: RecA-dependent chromosomal transformation and RecA-independent plasmid transformation require the competence-induced DprA mediator. Naturally transformable bacteria recombine internalized ssDNA with a homologous resident duplex (chromosomal transformation) or complementary internalized ssDNAs (plasmid or viral transformation). Bacillus subtilis competence-induced DprA, RecA, SsbB, and SsbA proteins are involved in the early processing of the internalized ssDNA, with DprA physically interacting with RecA. SsbB and SsbA bind and melt secondary structures in ssDNA but limit RecA loading onto ssDNA. DprA binds to ssDNA and facilitates partial dislodging of both single-stranded binding (SSB) proteins from ssDNA. In the absence of homologous duplex DNA, DprA does not significantly increase RecA nucleation onto protein-free ssDNA. DprA facilitates RecA nucleation and filament extension onto SsbB-coated or SsbB plus SsbA-coated ssDNA. DprA facilitates RecA-mediated DNA strand exchange in the presence of both SSB proteins. DprA, which plays a crucial role in plasmid transformation, anneals complementary strands preferentially coated by SsbB to form duplex circular plasmid molecules. Our results provide a mechanistic framework for conceptualizing the coordinated events modulated by SsbB in concert with SsbA and DprA that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.

Effect of the recU suppressors sms and subA on DNA repair and homologous recombination in Bacillus subtilis

Abstract. In Bacillus subtilis, mutant alleles of the genes sms and subA partially suppress the recombination phenotype of recU cells. When present in an otherwise Rec+ strain, Δsms and ΔsubA alleles render cells slightly sensitive to DNA-damaging agents, and impair chromosomal transformation (3- to 10-fold reduction), but do not affect plasmid transformation (less than 1.5-fold reduction). The Δsms and ΔsubA alleles were introduced into rec-deficient strains representative of the epistatic groups α (recF strain), β (addA addB), γ (recH), ε (recB, ΔrecU and recD strains) and ζ (ΔrecS). Both the Δsms and ΔsubA mutations were found to increase sensitivity to DNA-damaging agents in recF, ΔrecS and addAB cells. In contrast, the Δsms mutations decreased the sensitivity of recB, ΔrecU, recD and recH cells, and the ΔsubA mutation decreased the sensitivity of recB and ΔrecU cells to DNA-damaging agents. Functions classified within the epistatic groups α, ε and ζ are required for intramolecular recombination, measured as plasmid transformation. The Δsms and ΔsubA mutations, which partially suppressed the recombinational repair phenotype of mutants for functions within epistatic group ε, enhanced plasmid transformation of recU (recB, recD) and recS cells by 10- to 20-fold. In the absence of the proteins Sms and SubA, the recombination machinery is apparently redirected towards (an) alternative pathway(s). Furthermore, the shared ability of the Δsms and ΔsubA mutations to act as indirect suppressors of recB, recU and recD mutations supports the classification of the recBUD genes within epistatic group ε.

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.