Oligomeric Initiator Protein-Mediated DNA Looping Negatively Regulates Plasmid Replication In Vitro by Preventing Origin Melting

Zzaman, Shamsu; Bastia, Deepak

doi:10.1016/j.molcel.2005.10.037

Cited by 34 publications

(50 citation statements)

References 43 publications

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“…Indeed, in the crystal packing, ␣5 interacts with its symmetry counterpart, which tentatively suggests a coiled-coil mechanism of dimerization. Dimers of are repressors of replication (27,35), and dimer-dimer interactions involving two sets of iterons of a pair of plasmids in trans are known to shut down initiation by handcuffing. Alternatively, interaction of a single array of dimers binding to two sets of iterons and causing coupling or handcuffing of a pair of origins is believed to be a major mechanism of negative control of replication (19,36).…”

Section: Mutant Forms Ofmentioning

confidence: 99%

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Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

Swan¹,

Bastia²,

Davies³

2006

Proc. Natl. Acad. Sci. U.S.A.

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We have determined the crystal structure of a monomeric biologically active form of the initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-Å resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal ␣4 and the N-terminal ␣4 helices, that recognize the 5 half and the 3 half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in ␣4 , whereas the ␣4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely ␣2 and ␣5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.initiator structure ͉ replication control ͉ replication initiation I n a major class of drug resistance plasmids, interaction of a plasmid-encoded replication initiator protein with the originproximal, cognate, repeated sequences called iterons, initiates Cairns-type replication that generates -shaped replication intermediates (1-12). Although a wealth of information exists on the genetics and biochemistry of replication initiation and its control in a few model plasmid systems, there has been a paucity of information on the atomic structure of the plasmid-encoded replication initiator proteins and their detailed structurefunction analysis. In fact, the structure of only one plasmidencoded initiator, namely RepE of F factor, is known to date (13). Unfortunately, the F plasmid system offers very few mutants of the RepE initiator that have been isolated and analyzed to date and very little published information exists on the interaction of the RepE initiator with host-encoded replication proteins.By contrast, the R6K plasmid system has a rich repertoire of biological and biochemical information on its initiator protein called (2,3,6,8,(14)(15)(16)(17)(18)(19), including its interaction with several host-encoded replication proteins (20-22), and its self interaction, via dimerization (23, 24), dimer-dimer (or dimer-monomer) interactions, that are essential for DNA looping (22). The R6K replicons consist of three replication origins called ␣, ␤, and ␥. is a transacting initiator protein that binds to seven tandem iterons at ␥ (Fig. 1). Dimerization of negatively controls copy number, as revealed by the fact that mutations that enhance copy number also tend to monomerize the protein upon iteron binding (22,23,25). In addition to the dimerization interphase, a second type ofcontact is required for dimer-dimer or, more likel...

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Section: Mutant Forms Ofmentioning

confidence: 99%

“…1b). Alternatively, a dimeric could bridge pairs of iteron-bound monomers (35,38). The looping models suggest that, besides the dimerization interphase, a second type of protein-protein contact involving a dimer and a monomer or two dimers would be needed for DNA looping.…”

Section: Mutant Forms Ofmentioning

confidence: 99%

Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

Swan¹,

Bastia²,

Davies³

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Eukaryotic initiation factors such as Cdt1 are inactivated after replication initiation by one or more of several mechanisms, such as inhibitory phosphorylation, proteolysis, titration by an inhibitor (geminin), and exportation out of the nucleus (22).…”

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confidence: 99%

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Venkova-Canova¹,

Srivastava²,

Chattoraj³

2006

Proc. Natl. Acad. Sci. U.S.A.

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The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.O ur knowledge of DNA replication control in bacteria comes largely from studying the chromosome and plasmids of Escherichia coli (1, 2). In general, controlling proteins that initiate replication, initiator proteins, seems to be the central mechanism to regulate replication. A variety of mechanisms have been found to control the initiator.Transcriptional autoregulation of the initiator gene is common in plasmids with iterated initiator-binding sites (iterons), where the initiator serves as the repressor of its own synthesis (3). The E. coli initiator, DnaA, also is autoregulated (4). In addition, the dnaA promoter is inactivated by sequestration to the cell membrane for approximately one-third of the cell generation (5). More traditional modes of transcriptional control using repressors and activators also have been found in phage and plasmid RK2 (6, 7).Initiator translation is regulated by antisense RNAs in a variety of plasmids, the classic examples being plasmids R1 and pT181 (8, 9). The antisense RNA either blocks the ribosomebinding site of the initiator gene directly or of a regulatory peptide for the initiator gene (10).Most other mechanisms operate posttranslationally either by titrating or inactivating the initiator protein. In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.Initiator inactivation is the most widely used mechanism to prevent premature replication reinitiation. The pT181 initiator is inactivated by covalent modification at the end of one round of replication (13). E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14-16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Euk...

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“…A third mechanism that may underlie the difference in PCNs between TrfA1 and TrfA2 is the inhibition of oriV melting by blocking the replication origin by TrfA dimers that bridge iteron clusters on separate molecules (so-called handcuffing) (31) or iteron clusters on the same molecule (71). Although the polypeptides involved in the TrfA dimer formation seem to be localized in the C-terminal region of TrfA2 (9,64), it is unclear how TrfA monomers and dimers interact in the replication inhibition process.…”

Section: Discussionmentioning

confidence: 99%

Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

et al. 2012

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Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1␤ plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-␤ replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3-to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts. P lasmids belonging to incompatibility group P-1 (IncP-1 group) are generally considered to have a broad host range owing to their ability to replicate in at least two classes within the phylum Proteobacteria (e.g., Alphaproteobacteria and Gammaproteobacteria). The majority of IncP-1 plasmids have been identified as agents that mediate transfer of beneficial traits between these phylogenetically distinct bacteria, such as antibiotic resistance, resistance to heavy metals, or catabolic activity of xenobiotic compounds (2, 51, 60). Since the worldwide spread of antibiotic resistance is a growing concern in human health care (40), it is important that we improve our understanding of the mechanisms by which IncP-1 plasmids are maintained in a broad range of hosts.Plasmid RK2, identified in Pseudomonas aeruginosa, has been studied as a model IncP-1 plasmid to gain understanding of basic plasmid functions (i.e., replication, stable inheritance, and conjugative transfer) and of the promiscuous nature of the IncP-1 group (32). It is known that the replication initiation protein (trans-acting factor A, TrfA) and the replication origin (oriV) are responsible for RK2 replication in a broad range of hosts (20, 52). RK2 encodes two replication initiation proteins (Rep proteins), 56). TrfA-33 is the truncated version of TrfA-44 and is produced ...

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Oligomeric Initiator Protein-Mediated DNA Looping Negatively Regulates Plasmid Replication In Vitro by Preventing Origin Melting

Cited by 34 publications

References 43 publications

Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

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