A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.     

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

DNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ΔrecD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ΔrecD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5′-3′ helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.     

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.     

A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus

In Gram negative Escherichia coli there are two well-characterised primosomal assembly processes, the PriA- and DnaA-mediated cascades. The presence of PriA and DnaA proteins in Gram positive Bacillus spp. supports the assumption that both the PriA- and DnaA-mediated primosomal assembly cascades also operate in these organisms. However, the lack of sequence homology between the rest of the primosomal proteins indicates significant differences between these two bacterial species. Central to the process of primosomal assembly is the loading of the main hexameric replicative helicase (DnaB in E.coli and DnaC in Bacillus subtilis) on the DNA. This loading is achieved by specialised proteins known as ‘helicase loaders’. In E.coli DnaT and DnaC are responsible for loading DnaB onto the DNA during primosome assembly, in the PriA- and DnaA-mediated cascades, respectively. In Bacillus the identity of the helicase loader is still not established unequivocally. In this paper we provide evidence for a functional interaction between the primosomal protein DnaI from B.subtilis and the main hexameric replicative helicase DnaB from Bacillus stearothermophilus. Our results are consistent with the putative role of DnaI as the ‘helicase loader’ in the Gram positive Bacillus spp.     

Primosome assembly site in Bacillus subtilis.

A single-strand initiation site was detected on the Enterococcus faecalis plasmid pAM beta 1 by its ability to prevent accumulation of single stranded DNA of a rolling circle plasmid, both in Bacillus subtilis and Staphylococcus aureus. This site, designated ssiA, is located on the lagging strand template, approximately 150 bp downstream from the replication origin. ssiA priming activity requires the DnaE primase, the DnaC replication fork helicase, as well as the products of the dnaB, dnaD and dnaI genes of B.subtilis, but not the RNA polymerase. The primase and the replication fork helicase requirements indicate that ssiA is a primosome assembly site. Interestingly, the pAM beta 1 lagging strand synthesis is inefficient when any of the proteins involved in ssiA activity is mutated, but occurs efficiently in the absence of ssiA. This suggests that normal plasmid replication requires primosome assembly and that the primosome can assemble not only at ssiA but also elsewhere on the plasmid. This work for the first time describes a primosome in a Gram-positive bacterium. Involvement of the B.subtilis proteins DnaB, DnaD and DnaI, which do not have any known analogue in Escherichia coli, raises the possibility that primosome assembly and/or function in B.subtilis differs from that in E.coli.     

Localization of a cryptic binding site for tenascin on fibronectin

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.     

The transition state regulator Hpr of Bacillus subtilis is a DNA-binding protein

The synthesis of a variety of proteins, including the well characterized degradative enzymes, which occurs during the transition state between vegetative growth and the onset of sporulation in Bacillus subtilis is controlled by a class of molecules known as transition state regulators. One of these regulators is the product of the hpr gene, first identified by mutations affecting the synthesis of extracellular proteases. We have purified the Hpr protein and found that it binds specifically to DNA fragments carrying the promoters and the upstream regions of the alkaline (aprE) and neutral (nprE) protease genes of B. subtilis. DNase I protection experiments revealed that the Hpr protein is able to bind at four and two regions of the aprE and nprE promoters, respectively. We have also located two Hpr binding sites in the promoter region of a gene of unknown function which is nevertheless known to be developmentally regulated during the transition state and which occurs in the same operon as the gene encoding another transition state regulator, Sin. The location of one of the Hpr binding sites on the aprE gene occurs adjacent to a region to which the Sin protein binds. However, in mixing competition experiments we have shown that Hpr and Sin binding occurred independently, and no visible alterations of protected regions were detected.     

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA

Genetic evidence suggests that the Bacillus subtilis lrpC gene product participates in cell growth and sporulation. The purified LrpC protein, which has a predicted molecular mass of 16.4 kDa, is a tetramer in solution. LrpC binds with higher affinity (K_app ~ 80 nM) to intrinsically curved DNA than to non-curved DNA (K_app ~ 700 nM). DNase I footprinting and the supercoiling of relaxed circular plasmid DNA in the presence of topoisomerase I revealed that LrpC induces DNA bending and constrains DNA supercoils in vitro. The LrpC protein cooperatively increases DNA binding of the bona fide DNA-binding and DNA-bending protein Hbsu. LrpC forms inter- and intramolecular bridges on linear and supercoiled DNA molecules, resulting in a large network and DNA compactation. Collectively, these findings suggest that LrpC is an architectural protein and that its activities could provide a means to modulate DNA transactions.     

Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus

Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.     

FAD binding in glycine oxidase from Bacillus subtilis

The apoprotein of the FAD-containing flavoenzyme glycine oxidase from Bacillus subtilis was obtained at pH 8.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M Tris–HCl and 20% glycerol. The apoprotein of glycine oxidase shows high protein fluorescence, high exposure of hydrophobic surfaces, and low temperature stability as compared to the holoenzyme. The isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD. The reconstitution process follows a particular two-stage process; the spectral properties of the reconstituted holoenzyme were virtually indistinguishable from those observed with native glycine oxidase, while the activity was only partially (50%) recovered. The urea-induced unfolding process of glycine oxidase can be considered as a two-step (three-state) process: the presence of intermediate(s) in the unfolding process of the holoenzyme at ≈2 M urea is evident in the changes of the flavin fluorescence intensity and can be also inferred from the different urea sensitivities of the spectral probes used. On the other hand, only a single transition at ≈4.5 M urea concentration is observed for the apoprotein form. The chemical denaturation of glycine oxidase holoenzyme is partially reversible (e.g., no activity is recovered when starting the refolding from 4 M urea-denatured holoprotein). Finally, the introduction by site-directed mutagenesis of residues corresponding to those involved in the covalent link with FAD in the related flavoenzyme monomeric sarcosine oxidase failed to convert glycine oxidase into a covalent flavoprotein. These investigations show that the consequences of FAD binding for the stability and folding process distinguish glycine oxidase from enzymes active on similar compounds.     

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.     

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein.

In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA.     

Methanol production during chemotaxis to amino acids in Bacillus subtilis

The 20 common amino acids act as attractants during chemotaxis by the Gram-positive organism Bacillus subtilis . In this study, we report that all amino acids induce B. subtilis to produce methanol both upon addition and removal of the chemoeffector. Asparagine-induced methanol production is specific to the McpB receptor and aspartate-induced methanol production correlates with receptor occupancy. These findings suggest that addition and removal of all amino acids cause demethylation of specific receptors which results in methanol production. We also demonstrate that certain attractants cause greater production of methanol after multiple stimulations. CheC and CheD, while affecting the levels of receptor methylation, are not absolutely required for either methylation or demethylation. In contrast, CheY is necessary for methanol formation upon removal of attractant but not upon addition of attractant. We conclude that methanol formation due to negative stimuli indicates the existence of a unique adaptational mechanism in B. subtilis involving the response regulator, CheY.