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PubMed1997

Nicking by transesterification: the reaction catalysed by a relaxase.

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer. Purification and characterization of relaxases from several plasmids has revealed the reaction mechanism: relaxases nick duplex DNA in a site- and strand-specific manner by catalysing a transesterification. The product of the reaction is a nicked double-stranded DNA molecule with a sequestered 3'-OH and the relaxase covalently bound to the 5' end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic and reversible; a second transesterification ligates the nicked DNA. However, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediate in the process of conjugative DNA transfer. Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand of DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some, but not all, plasmid systems. The first relaxase-catalysed transesterification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the process. The relaxase, in conjunction with several auxiliary proteins, forms the relaxation complex or relaxosome first described nearly 30 years ago as being associated with conjugative and mobilizable plasmids.

Amino Acid Sequence↗

PubMed1997

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli.

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication (oriC) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC. GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis-acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.

Base Sequence↗

PubMed1997

FIS modulates growth phase-dependent topological transitions of DNA in Escherichia coli.

The Escherichia coli DNA-binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site-specific inversion of DNA and transcriptional activation of stable RNA promoters. In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes. We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA. However, FIS also binds and bends DNA at many non-specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E. coli chromosome. Here, we show that fis affects growth phase-specific alterations in the supercoiling level of DNA. Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift-up. In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state. Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth.

Carrier Proteins↗

PubMed1998

DNA-binding characteristics of the Escherichia coli CytR regulator: a relaxed spacing requirement between operator half-sites is provided by a flexible, unstructured interdomain linker.

The Escherichia coli CytR regulator belongs to the LacI family of sequence-specific DNA-binding proteins and prevents CRP-mediated transcription in the CytR regulon. Unlike the other members of this protein family, CytR binds with only modest affinity to its operators and transcription repression thus relies on the formation of nucleoprotein complexes with the cAMP-CRP complex. Moreover, CytR exhibits a rotational and translational flexibility in operator binding that is unprecedented in the LacI family. In this report we examined the effect of changing the spacing between CytR half-operators on CytR regulation in vivo and on CytR binding in vitro. Maximum repression was seen with the short spacing variants: repression peaks when the half-operators lie on the same face of the DNA helix. Repression was retained for most spacing variants with centre separations of half-operators < or = 3 helical turns. Our data confirm and extend the view that CytR is a highly flexible DNA binder that can adapt many different conformations for co-operative binding with CRP. Furthermore, limited proteolysis of radiolabelled CytR protein showed that the interdomain linker connecting the DNA binding domains and the core part of CytR does not become structured upon DNA binding. We conclude that CytR does not use hinge alpha-helices for minor groove recognition. Rather, CytR possesses a highly flexible interdomain linker that allows it to form complexes with CRP at promoters with quite different architecture.

Amino Acid Sequence↗

PubMed1999

VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium.

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.

Bacterial Proteins↗

PubMed2000

Plasmid and chromosome partitioning: surprises from phylogeny.

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par-encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter-cell positions). Two types of partitioning ATPases are known: the Walker-type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin-like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid-encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome-encoded ATPases, including members from both Gram-negative and Gram-positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.

Adenosine Triphosphatases↗

PubMed2000

The partition system of multidrug resistance plasmid TP228 includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily.

The segregational stability of bacterial, low-copy-number plasmids is promoted primarily by active partition. The plasmid-specified components of the prototypical P1 plasmid partition system consist of two proteins, ParA (44.3 kDa) and ParB (38.5 kDa), which, in conjunction with integration host factor, form a nucleoprotein complex at the plasmid partition site, parS. This complex is the probable substrate for the directed temporal and spatial intracellular movement of plasmids before cell division. The genetic organization of the partition cassette of the multidrug resistance plasmid TP228 differs markedly from that of the P1 paradigm. The TP228 system includes a novel member (ParF; 22.0 kDa) of the ParA superfamily of ATPases, of which the P1 ParA protein is the archetype. However, the ParF protein and its immediate relatives form a discrete subgroup of the ParA superfamily, which evolutionarily is more related to the MinD subgroup of cell division proteins than to ParA of P1. The TP228 and P1 partition modules differ further in that the former does not include a parB homologue, but does specify a protein (ParG; 8.6 kDa) unrelated to ParB. Homologues of the parF gene are widely disseminated on eubacterial genomes, suggesting that ParF-mediated partition may be a common mechanism by which plasmid segregational stability is achieved.

Amino Acid Sequence↗

PubMed2001

Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression.

Secretion of effector proteins in enteropathogenic Escherichia coli (EPEC) is mediated by a specialized type III secretion system whose components are encoded in the LEE1, LEE2 and LEE3 operons. Using cat transcriptional fusions and primer extension analysis, we determined that the LEE2 and LEE3 operons are expressed from two overlapping divergent promoters, whose expression is negatively regulated by flanking common upstream and downstream silencing regulatory sequences (SRS1 and SRS2). In the absence of either SRS1 or SRS2, expression of the LEE2 and LEE3 operons became independent of Ler, a positive regulatory protein encoded by the first gene of the LEE1 operon. Similarly, in the absence of the histone-like protein H-NS, expression from both promoters became Ler independent even if both SRSs were present. In addition, the efficient expression of both the LEE2 and the LEE3 promoters required PerC (BfpW), a protein coded by the third gene of the per (bfpTVW) locus, but only in the presence of the EAF plasmid. Our deletion analysis also showed that the negative regulation observed in the presence of ammonium or at temperatures above 37 degrees C (e.g. 40 degrees C) required the SRSs or elements located therein. In contrast, the negative regulation observed in LB or at temperatures below 37 degrees C (e.g. 25 degrees C) was still observed even in the absence of both SRSs and seems to act only on the promoters. Together, these results suggest that Ler acts as an antirepressor protein that overcomes the H-NS-mediated silencing on the LEE2/LEE3 divergent promoter region, which is probably caused by the formation of a repressing H-NS-nucleoprotein complex.

Amino Acid Sequence↗

PubMed2001

The Escherichia coli histone-like protein HU regulates rpoS translation.

Escherichia coli HU protein is a major component of the bacterial nucleoid. HU stabilizes higher order nucleoprotein complexes and belongs to a family of DNA architectural proteins. Here, we report that HU is required for efficient expression of the sigma S subunit of RNA polymerase. This rpoS-encoded alternative sigmaS factor induces a number of genes implicated in cell survival in stationary phase and in multiple stress resistance. By analysis of rpoS-lacZ fusions and by pulse-chase experiments, we show that the efficiency of rpoS translation is reduced in cells lacking HU, whereas neither rpoS transcription nor protein stability is affected by HU. Gel mobility shift assays show that HU is able to bind specifically an RNA fragment containing the translational initiation region of rpoS mRNA 1000-fold more strongly than double-stranded DNA. Together with the in vivo data, this finding strongly suggests that, by binding to rpoS mRNA, HU directly stimulates rpoS translation. We demonstrate here that HU, an abundant DNA-binding, histone-like protein, is able specifically to recognize an RNA molecule and therefore play a role in post-transcriptional regulation.

Bacterial Proteins↗

PubMed2001

Feedback controls restrain the initiation of Escherichia coli chromosomal replication.

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.

Bacterial Proteins↗

PubMed2001

A molecular mechanism for the repression of transcription by the H-NS protein.

The H-NS protein is a major component of the bacterial nucleoid and plays a crucial role in the global gene regulation of enteric bacteria. Although H-NS does not exhibit a high DNA sequence specificity, a number of H-NS-responsive promoters have been shown to contain regions of intrinsic DNA curvature located either upstream or downstream of the transcription start point. We have studied H-NS binding to DNA and in vitro transcriptional regulation by H-NS at several synthetic promoters with or without curved sequences inserted upstream of the Pribnow box. We show how such inserts determine the final organization of H-NS-containing nucleoprotein complexes and how this affects transcription. We refine a two-step mechanism for the constitution of H-NS assemblies that are efficient in regulation.

Bacterial Proteins↗

PubMed2003

The molecular basis of co-operative DNA binding between lambda integrase and excisionase.

Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.

Attachment Sites, Microbiological↗

PubMed2004

Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability.

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.

Animals↗

PubMed1994

The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility.

The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail. The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding. They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles. The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.

Base Sequence↗

PubMed2005

[Telomere and cancer: what's more at the end?].

Telomeres are nucleoprotein complexes that cap the end of eukaryotic chromosomes. They are essential for the functions and the stability of the genomes. There is now compelling evidences that telomerase, the enzyme that adds telomeric DNA repeats to chromosome end, is an important player in oncogenesis. The absence of telomerase in somatic tissues is thought to promote genome instability at initial stages of oncogenesis, favoring the emergence of cancer-associated chromosomal abnormalities \; restablishment of telomerase activity is expected afterwards if long term cell cycling is to occur. In addition to telomerase, various factors control the structure and function of telomeres, suggesting that additional telomeric components play important roles during oncogenesis.

Chromosomes↗

PubMed2005

Telomere length regulation during cloning, embryogenesis and ageing.

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes with an essential role in chromosome capping. Owing to the end-replication problem of DNA polymerase, telomeres shorten during each cell division. When telomeres become critically short, they loose their capping function, which in turn induces a DNA damage-like response. This mechanism inhibits cell proliferation at the senescence stage and there is evidence that it limits the regenerative capacity of tissues and organs during chronic diseases and ageing. The holoenzyme telomerase synthesises telomeric DNA de novo, but, in humans, it is active only during embryogenesis, in immature germ cells and in a subset of stem/progenitor cells during postnatal life. Telomere length can be maintained or increased by telomerase, a process that appears to be regulated by a variety of telomere-binding proteins that control telomerase recruitment and activity at the telomeres. During embryogenesis, telomerase is strongly activated at the morula/blastocyst transition. At this transition, telomeres are significantly elongated in murine and bovine embryos. Early embryonic telomere elongation is telomerase dependent and leads to a rejuvenation of telomeres in cloned bovine embryos. Understanding of the molecular mechanisms underlying this early embryonic telomere elongation programme is of great interest for medical research in the fields of regeneration, cell therapies and therapeutic cloning.

Aging↗

PubMed2005

TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase.

Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwBDeltaN70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwBDeltaN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwBDeltaN70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwBDeltaN70-DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor.

Adenosine Triphosphatases↗

PubMed2005

Bacterial DNA segregation by dynamic SopA polymers.

Many bacterial plasmids and chromosomes rely on ParA ATPases for proper positioning within the cell and for efficient segregation to daughter cells. Here we demonstrate that the F-plasmid-partitioning protein SopA polymerizes into filaments in an ATP-dependent manner in vitro, and that the filaments elongate at a rate that is similar to that of plasmid separation in vivo. We show that SopA is a dynamic protein within the cell, undergoing cycles of polymerization and depolymerization, and shuttling back and forth between nucleoprotein complexes that are composed of the SopB protein bound to sopC-containing plasmids (SopB/sopC). The dynamic behavior of SopA is critical for Sop-mediated plasmid DNA segregation; mutations that lock SopA into a static polymer in the cell inhibit plasmid segregation. We show that SopA colocalizes with SopB/sopC in the cell and that SopB/sopC nucleates the assembly of SopA and is required for its dynamic behavior. When SopA is polymerized in vitro in the presence of SopB and sopC-containing DNA, SopA filaments emanate from the plasmid DNA in radial asters. We propose a mechanism in which plasmid separation is driven by the polymerization of SopA, and we speculate that the radial assembly of SopA polymers is responsible for positioning plasmids both before and after segregation.

Bacterial Proteins↗