PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA.

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.

The tubulin-like RepX protein encoded by the pXO1 plasmid forms polymers in vivo in Bacillus anthracis.

Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.

Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time.

Many questions in molecular and cellular biology can be reduced to questions about 'who talks to whom, when and how frequently'. Here, we review approaches we have used with single-pair fluorescence resonance energy transfer (spFRET) to follow the motions between two well-placed fluorescent probes to ask similar questions. We describe two systems. We have used a nucleosomal system in which the naked DNA molecule has the acceptor and donor dyes too far apart for FRET to occur whereas the dyes are close enough in the reconstituted nucleosome for FRET. As these individual nucleosomes were tethered on a surface, we could follow dynamics in the repositioning of these two dyes, inferring that nucleosomes stochastically and reversibly open and close. These results imply that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix. In the case of following the binding of recombination protein RecA to double-stranded DNA (dsDNA) and the RecA filament displacement by DNA helicase motor PcrA, the dsDNA template is prepared with the two dyes close enough to each other to generate high FRET. Binding of the RecA molecules to form a filament lengthens the dsDNA molecule 1.5-fold and reduces the FRET accordingly. Once added, DNA motor protein helicase PcrA can displace the RecA filament with concomitant return of the DNA molecule to its original B-form and high FRET state. Thus, appropriately placed fluorescent dyes can be used to monitor conformational changes occurring in DNA and or proteins and provide increased sensitivity for investigating dynamic DNA-protein interactions in real time.

GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.

RepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTP-dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPgammaS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPgammaS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl-terminal region and exhibits GTP-dependent, non-specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.

DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange.

PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities.

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein-protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.

Structure-specific DNA binding and bipolar helicase activities of PcrA.

PcrA is an essential helicase in Gram-positive bacteria, but its precise role in cellular DNA metabolism is currently unknown. The Staphylococcus aureus PcrA helicase has both 5'-->3' and 3'-->5' helicase activities. In this work, we have studied the binding of S.aureus PcrA to a variety of DNA substrates that represent intermediates in DNA replication, repair, recombination and transcription. PcrA bound poorly or not at all to single-stranded DNA, double-stranded DNA with blunt ends, partially double-stranded DNA containing fork and bubble structures, and duplex DNA substrates containing either 5' or 3' single-stranded oligo dT tails. Interestingly, PcrA bound with high affinity to partially duplex DNA containing hairpin structures adjacent to a 6 nt long 5' single-stranded region and one unpaired nucleotide (flap) at the 3' end. However, PcrA did not detectably bind to partial duplexes with folded regions adjacent to a 6 nt long 3' single-stranded tail (with or without a 1 nt flap at the 5' end). PcrA showed moderate helicase activity with partially double-stranded DNAs containing 3' or 5' single-stranded oligo dT tails, the 3'-->5' helicase activity being more efficient than its 5'-->3' helicase activity. Interestingly, PcrA showed maximal helicase activity with substrates containing folded structures and 5' single-stranded tails, suggesting that its 5'-->3' helicase activity is greatly stimulated in the presence of specific structures. However, the 3'-->5' helicase activity of PcrA did not appear to be affected by the presence of folded substrates containing 3' single-stranded tails. Our data indicate that PcrA may recognize DNA substrates with specific structures in vivo and its 5'-->3' and 3'-->5' helicase activities may be involved in distinct cellular processes.

Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus.

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid rolling circle replication.

Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the rolling circle replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5' --> 3' helicase and had a weaker 3' --> 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.