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1.
Protein function often requires remodeling of protein structure. In the well-studied iteron-containing plasmids, the initiator of replication has a dimerization interface that undergoes chaperone-mediated remodeling. This remodeling reduces dimerization and promotes DNA replication, since only monomers bind origin DNA. A structurally homologs interface exists in RctB, the replication initiator of Vibrio cholerae chromosome 2 (Chr2). Chaperones also promote Chr2 replication, although both monomers and dimers of RctB bind to origin, and chaperones increase the binding of both. Here we report how five changes in the dimerization interface of RctB affect the protein. The mutants are variously defective in dimerization, more active as initiator, and except in one case, unresponsive to chaperone (DnaJ). The results indicate that chaperones also reduce RctB dimerization and support the proposal that the paradoxical chaperone-promoted dimer binding likely represents sequential binding of monomers on DNA. RctB is also activated for replication initiation upon binding to a DNA site, crtS, and three of the mutants are also unresponsive to crtS. This suggests that crtS, like chaperones, reduces dimerization, but additional evidence suggests that the remodelling activities function independently. Involvement of two remodelers in reducing dimerization signifies the importance of dimerization in limiting Chr2 replication.  相似文献   

2.
The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.  相似文献   

3.
Vibrio cholerae, the pathogenic bacterium that causes cholera, has two chromosomes (Chr1, Chr2) that replicate in a well-orchestrated sequence. Chr2 initiation is triggered only after the replication of the crtS site on Chr1. The initiator of Chr2 replication, RctB, displays activities corresponding with its different binding sites: initiator at the iteron sites, repressor at the 39m sites, and trigger at the crtS site. The mechanism by which RctB relays the signal to initiate Chr2 replication from crtS is not well-understood. In this study, we provide new insights into how Chr2 replication initiation is regulated by crtS via RctB. We show that crtS (on Chr1) acts as an anti-inhibitory site by preventing 39m sites (on Chr2) from repressing initiation. The competition between these two sites for RctB binding is explained by the fact that RctB interacts with crtS and 39m via the same DNA-binding surface. We further show that the extreme C-terminal tail of RctB, essential for RctB self-interaction, is crucial for the control exerted by crtS. This subregion of RctB is conserved in all Vibrio, but absent in other Rep-like initiators. Hence, the coordinated replication of both chromosomes likely results from the acquisition of this unique domain by RctB.  相似文献   

4.
H Komori  F Matsunaga  Y Higuchi  M Ishiai  C Wada    K Miki 《The EMBO journal》1999,18(17):4597-4607
The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N- and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.  相似文献   

5.
The region responsible for replication of Vibrio cholerae chromosome II (chrII) resembles those of plasmids that have repeated initiator binding sites (iterons) and an autorepressed initiator gene. ChrII has additional features: Its iterons require full methylation for initiator (RctB) binding, which makes them inactive for a part of the cell cycle when they are hemi-methylated. RctB also binds to a second kind of site, called 39-mers, in a methylation independent manner. This binding is inhibitory to chrII replication. The site that RctB uses for autorepression has not been identified. Here we show that a 29-mer sequence, similar to the 39-mers, serves as that site, as we find that it binds RctB in vitro and suffices to repress the rctB promoter in vivo. The site is not subject to methylation and is likely to be active throughout the cell cycle. The 29-mer, like the 39-mers, could inhibit RctB-dependent mini-chrII replication in Escherichia coli, possibly by coupling with iterons via RctB bridges, as was seen in vitro. The 29-mer thus appears to play a dual role in regulating chrII replication: one independent of the cell cycle, the other dependent upon iteron methylation, hence responsive to the cell cycle.  相似文献   

6.
Dam methylates GATC sequences in γ‐proteobacteria genomes, regulating several cellular functions including replication. In Vibrio cholerae, which has two chromosomes, Dam is essential for viability, owing to its role in chr2 replication initiation. In this study, we isolated spontaneous mutants of V. cholerae that were able to survive the deletion of dam. In these mutants, homologous recombination and chromosome dimer resolution are essential, unless DNA mismatch repair is inactivated. Furthermore, the initiator of chr2 replication, RctB, is no longer required. We show that, instead, replication of chr2 is insured by spontaneous fusion with chr1 and piggybacking its replication machinery. We report that natural fusion of chr1 and chr2 occurred by two distinct recombination pathways: homologous recombination between repeated IS elements and site‐specific recombination between dif sites. Lastly, we observed a preferential fusion of the two chromosomes in their terminus of replication.  相似文献   

7.
Koch B  Ma X  Løbner-Olesen A 《Plasmid》2012,68(3):159-169
RctB serves as the initiator protein for replication from oriCII, the origin of replication of Vibrio cholerae chromosome II. RctB is conserved between members of Vibrionaceae but shows no homology to known replication initiator proteins and has no recognizable sequence motifs. We used an oriCII based minichromosome to isolate copy-up mutants in Escherichia coli. Three point mutations rctB(R269H), rctB(L439H) and rctB(Y381N) and one IS10 insertion in the 3'-end of the rctB gene were obtained. We determined the maximal C-terminal deletion that still gave rise to a functional RctB protein to be 165 amino acids. All rctB mutations led to decreased RctB-RctB interaction indicating that the monomer is the active form of the initiator protein. All mutations also showed various defects in rctB autoregulation. Loss of the C-terminal part of RctB led to overinitiation by reducing binding of RctB to both rctA and inc regions that normally serve to limit initiation from oriCII. Overproduction of RctB(R269H) and RctB(L439H) led to a rapid increase in oriCII copy number. This suggests that the initiator function of the two mutant proteins is increased relative to the wild-type.  相似文献   

8.
Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.  相似文献   

9.
Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.  相似文献   

10.
Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.  相似文献   

11.
Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII. DnaA has binding sites at the chrI origin of replication as well as outside the origin. RctB likewise binds at the chrII origin and, as shown here, to external sites. The binding to the external sites in chrII inhibits chrII replication. A new kind of site was found in chrI that enhances chrII replication. Consistent with its enhancing activity, the chrI site increased RctB binding to those chrII origin sites that stimulate replication and decreased binding to other sites that inhibit replication. The differential effect on binding suggests that the new site remodels RctB. The chaperone-like activity of the site is supported by the finding that it could relieve the dependence of chrII replication on chaperone proteins DnaJ and DnaK. The presence of a site in chrI that specifically controls chrII replication suggests a mechanism for communication between the two chromosomes for replication.  相似文献   

12.
Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.  相似文献   

13.
RepA is the DNA replication initiator protein of the Pseudomonas plasmid pPS10. RepA has a dual function: as a dimer, it binds to an inversely-repeated sequence acting as a repressor of its own synthesis; as a monomer, RepA binds to four directly-repeated sequences to constitute a specialized nucleoprotein complex responsible for the initiation of DNA replication. We have previously shown that a Leucine Zipper-like motif (LZ) at the N-terminus of RepA is responsible for protein dimerization. In this paper we characterize the existence in RepA of two protein globular domains C-terminal to the LZ. We propose that dissociation of RepA dimers into monomers results in a conformational change from a compact arrangement of both domains, competent for binding to the operator, to an extended species that is suited for iteron binding. This model establishes the structural basis for the activation of DNA replication initiators in plasmids from Gram-negative bacteria.  相似文献   

14.
Escherichia coli chaperones DnaJ, DnaK and GrpE increase P1 plasmid initiator binding to the origin by promoting initiator folding. The binding allows initiation and also promotes pairing of origins which is believed to control initiation frequency. Chaperone-independent DNA binding mutants are often defective in replication control. We show here that these mutants have increased rates of association for DNA binding and defects in origin pairing. The increases in association rates were found to be due either to increased protein folding into active forms or to increases in the association rate constant, kon. Since the dissociation rate constants for DNA release with these mutants are not changed, it is unlikely that the DNA binding domain is affected. The pairing domain may thus control replication and modulate DNA binding. The role of the pairing domain in DNA binding can be significant in vivo as the selection for chaperone-independent binding favors pairing-defective mutants.  相似文献   

15.
The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.  相似文献   

16.
D S Hwang  A Kornberg 《Cell》1990,63(2):325-331
A sequence of three tandem repeats of a 13-mer in the replication origin (oriC) of E. coli is the highly conserved site of opening of the duplex for initiation of DNA synthesis. A protein that binds this sequence has been discovered in E. coli and purified to homogeneity. This novel 33 kd polypeptide behaves as a dimer. Binding to the 13-mers is specific and limited to this region. At a ratio of 10-20 monomers per oriC plasmid, the binding blocks initiation by preventing the opening of the 13-mer region by dnaA protein. Once the 13-mers are opened by dnaA protein action, the 33 kd protein is without effect on the subsequent stages of replication. The specificity of binding and profound inhibitory effect suggest a regulatory role for this protein at an early stage of chromosome initiation.  相似文献   

17.
DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.  相似文献   

18.
19.
There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains--one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.  相似文献   

20.
Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.  相似文献   

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