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Dhruba K. Chattoraj - One of the best experts on this subject based on the ideXlab platform.
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The newly identified chrII sites inhibit mini-chrII replication in E. coli and bind purified RctB.
2014Co-Authors: Jong Hwan Baek, Dhruba K. ChattorajAbstract:(A) The activity of the chrII origin, oriII, was tested using a three-plasmid system in E. coli, where one plasmid carried oriII, poriII (pTVC35), another supplied RctB under arabinose control (pTVC11) and the third was either the empty vector (vector; pTVC243) or carried one of the new sites. As positive controls we used plasmids carried an Iteron (pIteron; pBH127) or a 39-mer (p39-mer; pTVC222). RctB was supplied at low (light gray bar) and high (dark gray bar) concentrations, using arabinose at 0.002% and 0.2%, respectively. The copy numbers of poriII were normalized to the copy number of poriII (called 1) when the third plasmid was the empty vector (pTVC243) and arabinose was at 0.002%. The mean values and standard deviations are from three independent experiments. (B) Binding of purified RctB to the new sites was tested by EMSA. Percent binding ([intensity of the retarded band/combined intensities of retarded and free bands]×100) at 2 nM (+) or 20 nM (++) are shown by light and dark gray bars, respectively. The error bars are from three independent measurements of band intensities from the same gel.
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Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer
Nucleic acids research, 2012Co-Authors: Jyoti K. Jha, Tatiana Venkova-canova, Gaëlle Demarre, Dhruba K. ChattorajAbstract: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.
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A 29-mer site regulates transcription of the initiator gene as well as function of the replication origin of Vibrio cholerae chromosome II.
Plasmid, 2012Co-Authors: Tatiana Venkova-canova, Anik Saha, Dhruba K. ChattorajAbstract: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.
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Transition from a plasmid to a chromosomal mode of replication entails additional regulators
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Tatiana Venkova-canova, Dhruba K. ChattorajAbstract:Plasmid origins of replication are rare in bacterial chromosomes, except in multichromosome bacteria. The replication origin of Vibrio cholerae chromosome II (chrII) closely resembles Iteron-bearing plasmid origins. Iterons are repeated initiator binding sites in plasmid origins and participate both in replication initiation and its control. The control is mediated primarily by coupling of Iterons via the bound initiators ("handcuffing"), which causes steric hindrance to the origin. The control in chrII must be different, since the timing of its replication is cell cycle-specific, whereas in plasmids it is random. Here we show that chrII uses, in addition to Iterons, another kind of initiator binding site, named 39-mers. The 39-mers confer stringent control by increasing handcuffing of Iterons, presumably via initiator remodeling. Iterons, although potential inhibitors of replication themselves, restrain the 39-mer-mediated inhibition, possibly by direct coupling ("heterohandcuffing"). We propose that the presumptive transition of a plasmid to a chromosomal mode of control requires additional regulators to increase the stringency of control, and as will be discussed, to gain the capacity to modulate the effectiveness of the regulators at different stages of the cell cycle.
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Origin pairing ('handcuffing') and unpairing in the control of P1 plasmid replication.
Molecular microbiology, 2004Co-Authors: Nilangshu Das, Dhruba K. ChattorajAbstract:The P1 plasmid origin has an array of five binding sites (Iterons) for the plasmid-encoded initiator protein RepA. Saturation of these sites is required for initiation. Iterons can also pair via their bound RepAs. The reaction, called handcuffing, is believed to be the key to control initiation negatively. Here we have determined some of the mechanistic details of the reaction. We show that handcuffed RepA-Iteron complexes dissociate when they are diluted or challenged with cold competitor Iterons, suggesting spontaneous reversibility of the handcuffing reaction. The complex formation increases with increased RepA binding, but decreases upon saturation of binding. Complex formation also decreases in the presence of molecular chaperones (DnaK and DnaJ) that convert RepA dimers to monomers. This indicates that dimers participate in handcuffing, and that chaperones are involved in reversing handcuffing. They could play a direct role by reducing dimers and an indirect role by increasing monomers that would compete out the weaker binding dimers from the origin. We propose that an increased monomer to dimer ratio is the key to reverse handcuffing.
K. Veluthambi - One of the best experts on this subject based on the ideXlab platform.
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Infectivity analysis of two variable DNA B components ofMungbean yellow mosaic virus-Vigna inVigna mungo andVigna radiata
Journal of Biosciences, 2004Co-Authors: V. Balaji, R. Vanitharani, A. S. Karthikeyan, S. Anbalagan, K. VeluthambiAbstract:Mungbean yellow mosaic virus-Vigna (MYMV-Vig), a Begomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram ( Vigna mungo ). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) Iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first Iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first Iteron besides having an 18-nt insertion between the third Iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram ( V. mungo ) and mungbean ( V. radiata ) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range between V. mungo and V. radiata .
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Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vigna in Vigna mungo and Vigna radiata.
Journal of biosciences, 2004Co-Authors: V. Balaji, R. Vanitharani, A. S. Karthikeyan, S. Anbalagan, K. VeluthambiAbstract:Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) Iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first Iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first Iteron besides having an 18-nt insertion between the third Iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.
Donald R. Helinski - One of the best experts on this subject based on the ideXlab platform.
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The Plasmid RK2 Initiation Protein Binds to the Origin of Replication as a Monomer
The Journal of biological chemistry, 1996Co-Authors: Aresa Toukdarian, Donald R. Helinski, Silvia PerriAbstract:Abstract The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (Iterons) present at the plasmid replication origin to initiate DNA replication. Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo. Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation. The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the Iteron DNA as a monomer. Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity. These results indicate that only the monomer form of the TrfA protein is active for binding to the Iterons at the RK2 replication origin.
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Isolation and Characterization of DNA-Binding Mutants of a Plasmid Replication Initiation Protein Utilizing an in Vivo Binding Assay
Plasmid, 1994Co-Authors: Joan Lin Cereghino, Donald R. Helinski, Aresa ToukdarianAbstract:Abstract An in vivo screen was developed for the identification of mutants of the RK2 replication initiation protein, TrfA, that were altered in their binding to the Iterons within the plasmid RK2 origin of replication. This assay is based on an antibiotic selection system originally described by Elledge, Sugiono, Guarente, and Davis (Proc. Natl. Acad. Sci. USA86, 3689-3693, 1989) for the isolation in vivo of genes encoding sequence-specific DNA-binding proteins. A TrfA-specific binding site consisting of two 17-bp Iterons separated by a nonrandom 6-bp spacer was placed 3 to a strong constitutive promoter. This promoter-Iteron fragment was then inserted into the assay vector convergent to the aadA gene such that an increased level of spectinomycin resistance by the Escherichia coli host was dependent on the binding of wild-type TrfA protein to the binding site. The in vivo system was used to specifically isolate TrfA mutants which were either defective in binding or capable of effecting increased levels of spectinomycin resistance as compared to wild-type TrfA. The defective TrfA mutants isolated by this screen were purified and found to be considerably less effective in DNA binding by in vitro gel mobility shift assays. The map location was determined for these six defective TrfA mutants. Each of the mutations consisted of a single base change and mapped within codons extending over a 162 amino acid sequence. All of the mutants which were capable of effecting increased levels of spectinomycin resistance in the in vivo DNA-binding assay also showed some alteration in RK2 replication in vivo with most of the mutants having a copy-up phenotype similar to previously isolated TrfA mutants able to maintain an eight-Iteron RK2 origin plasmid at a higher copy number.
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Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria.
The Journal of biological chemistry, 1993Co-Authors: Joan Lin Cereghino, Donald R. HelinskiAbstract:In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria. In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions. Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the Iterons at the replication origin. Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria. Substitution of a serine at that position preserved limited function in replication and DNA binding. The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function. Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin Iterons but caused a complete loss of replication activity in all four bacteria. Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting Iteron binding activity. However, substitution of this arginine with a lysine resulted in a loss of replication activity in E. coli and A. vinelandii but had no effect in P. putida and A. tumefaciens. These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its Iteron binding activity.
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DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats.
The Journal of biological chemistry, 1993Co-Authors: S Perri, Donald R. HelinskiAbstract:Abstract Replication of plasmid RK2 in a variety of Gram-negative bacteria requires its origin of replication and the plasmid-encoded TrfA proteins (TrfA-33 and TrfA-44). The initiation of replication requires that the TrfA proteins bind to a series of 17-base pair (bp) direct repeats located within the RK2 origin. The conserved 17-bp repeats are arranged in tandem and are separated by less conserved spacer sequences of 4-6 bp in length. A series of plasmids containing one or two Iterons, with or without the less conserved spacer sequences, were constructed to analyze the DNA sequence requirements for binding of TrfA-33 to the Iterons. In addition to the analysis of TrfA binding in vitro, the plasmid constructs were examined for their ability to exert incompatibility toward an RK2 replicon in Escherichia coli. These analyses revealed that the conserved 17-bp Iteron sequence itself is not sufficient for TrfA binding; the adjacent less conserved spacer sequences are also required. Site-specific mutagenesis was carried out to determine the importance of specific bases within the spacer sequence for binding activity and a consensus sequence for a TrfA-specific binding unit was determined. DNase I and methylation interference footprinting procedures were also carried out to characterize the TrfA-binding unit complex. Finally, it was shown that the binding of the TrfA-33 protein to two adjacent TrfA binding units on a DNA fragment is not substantially affected by the relative orientation or spacing between the two units.
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Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.
The Journal of biological chemistry, 1991Co-Authors: S Perri, Donald R. Helinski, Aresa ToukdarianAbstract:The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (Iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five Iterons was identical for all three protein preparations and extended from the beginning of the first Iteron to 5 base pairs upstream of the fifth Iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five Iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two Iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five Iterons were measured. This analysis showed that unlike complexes involving two or five Iterons the TrfA/one Iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or Iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the Iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.
Marcin Filutowicz - One of the best experts on this subject based on the ideXlab platform.
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cooperative binding mode of the inhibitors of r6k replication π dimers
Journal of Molecular Biology, 2008Co-Authors: Lisa M. Bowers, Marcin FilutowiczAbstract:Abstract The replication initiator protein, π, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of π bind to Iterons in the γ origin of plasmid R6K, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. Consequently, π monomers activate replication, while π dimers inhibit replication. Recently, it was shown that the monomeric form of π binds multiple tandem Iterons in a strongly cooperative fashion, which might explain how monomers outcompete dimers for replication initiation when plasmid copy number and π supply are low. Here, we examine cooperative binding of π dimers and explore the role that these interactions may have in the inactivation of γ origin. To examine π dimer/Iteron interactions in the absence of competing π monomer/Iteron interactions using wild-type π, constructs were made with key base changes to each Iteron that eliminate π monomer binding yet have no impact on π dimer binding. Our results indicate that, in the absence of π monomers, π dimers bind with greater cooperativity to alternate Iterons than to adjacent Iterons, thus preferentially leaving intervening Iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by π dimers.
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Mechanism of Origin Activation by Monomers of R6K-encoded π Protein
Journal of molecular biology, 2007Co-Authors: Lisa M. Bowers, Ricardo Krüger, Marcin FilutowiczAbstract:One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called Iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem Iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to Iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for Iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent Iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two Iterons but is diminished only slightly by insertion of a full helical turn between two Iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/Iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-Iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/Iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem Iterons.
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NOTES Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the �/DNA Interface �
2007Co-Authors: Selvi Kunnimalaiyaan, Sheryl A. Rakowski, Marcin FilutowiczAbstract:In previous work, we characterized the bases in an Iteron of plasmid R6K that are important for the binding of � protein monomers and dimers. Here we investigate the following six amino acids of �, encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254. Rep proteins activate replication origins (ori’s) by binding to tandem repeats in the DNA called Iterons (3, 8, 13). When the sequences of Iterons and rep genes are compared across families, striking similarities are observed (2, 10, 16–18). A breakthrough in Rep/Iteron studies came when Komori et al. published the crystal structure of a RepE monomer (from the F plasmid) in complex with its Iteron DNA (10). The structure revealed that the Rep monomer contains two DNA-binding domains, an N-terminal WH1 (winged helix 1) and a C-terminal WH2 domain, with both domains used for Iteron binding. A sequence alignment of several Reps presented by our group (10, 13) is a useful reference for identifying possible conserve
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Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons
Journal of bacteriology, 2005Co-Authors: Selvi Kunnimalaiyaan, Sheryl A. Rakowski, Ross B. Inman, Marcin FilutowiczAbstract:One proposed mechanism of replication inhibition in Iteron-containing plasmids (ICPs) is “handcuffing,” in which the coupling of origins via Iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded π protein with the seven 22-bp Iterons of the γ origin of replication. Like other related Rep proteins, π exists as both monomers and dimers. However, the ability of π dimers to bind Iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind Iterons. Here, we describe experiments to determine if monomers or dimers of π protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using π variants with different propensities to bind Iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (π·P106L∧F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of π protein (π·M36A∧M38A), which lacks initiator function, handcuffs Iteron-containing DNA more efficiently than does wild-type π. The data suggest that π dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the γ ori system. Thus, dimers of π appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of π expression, in cis competition (for origin binding) with monomeric activator π, and handcuffing-mediated inhibition of replication in trans.
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Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein π to the γ ori Iteron of Plasmid R6K
The Journal of biological chemistry, 2004Co-Authors: Selvi Kunnimalaiyaan, Ricardo Krüger, Sheryl A. Rakowski, Wilma Ross, Marcin FilutowiczAbstract:Abstract Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the γ origin of plasmid R6K, the Rep protein, π, is distinctive in that it can bind the seven 22-bp Iterons in two forms; π monomers activate replication, whereas π dimers act as inhibitors. In this work, we used wild type and variants of the π protein with altered monomer/dimer ratios to study Iteron/π 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 π monomers and dimers. We also isolated Iteron mutants that affected the binding of π monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, π monomers interact with nucleotides spanning the entire length of the Iteron. In contrast, π 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.
Rafael Giraldo - One of the best experts on this subject based on the ideXlab platform.
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Fluorescence studies of the replication initiator protein RepA in complex with operator and Iteron sequences and free in solution.
The FEBS journal, 2008Co-Authors: Rutger E. M. Diederix, Rafael Giraldo, Cristina Dávila, M. Pilar LilloAbstract:RepA, the replication initiator protein from the Pseudomonas plasmid pPS10, regulates plasmid replication and copy number. It is capable of autorepression, in which case it binds as a dimer to the inverted repeat operator sequence preceding its own gene. RepA initiates plasmid replication by binding as a monomer to a series of four adjacent Iterons, which contain the same half-repeat as found in the operator sequence. RepA contains two domains, one of which binds specifically to the half-repeat. The other is the dimerization domain, which is involved in protein–protein interactions in the dimeric RepA–operon complex, but which actually binds DNA in the monomeric RepA–Iteron complex. Here, detailed fluorescence studies on RepA and an N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-labeled single-cysteine mutant of RepA (Cys160) are described. Using time-resolved fluorescence depolarization measurements, the global rotational correlation times of RepA free in solution and bound to the operator and to two distinct Iteron dsDNA oligonucleotides were determined. These provide indications that, in addition to the monomeric RepA–Iteron complex, a stable dimeric RepA–Iteron complex can also exist. Further, Forster resonance energy transfer between Trp94, located in the dimerization domain, and N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-Cys160, located on the DNA-binding domain, is observed and used to estimate the distance between the two fluorophores. This distance may serve as an indicator of the orientation between both domains in the unbound protein and RepA bound to the various cognate DNA sequences. No major change in distance is observed and this is taken as evidence for little to no re-orientation of both domains upon complex formation.
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Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers
Molecular microbiology, 2008Co-Authors: Fátima Gasset-rosa, M. Elena Fernández-tresguerres, Teresa Díaz-lópez, Rudi Lurz, Alicia Prieto, Rafael GiraldoAbstract:In many plasmid replicons of gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (Iterons) or chaperones. Rep proteins play another key role: they bridge together two Iteron DNA stretches, found either on the same or on different plasmid molecules. These so-called, respectively, 'looped' and 'handcuffed' complexes are thought to be negative regulators of plasmid replication. Although evidence for Rep-dependent plasmid handcuffing has been found in a number of replicons, the structure of these Rep-DNA assemblies is still unknown. Here, by a combination of proteomics, electron microscopy, genetic analysis and modelling, we provide insight on a possible three-dimensional structure for two handcuffed arrays of the Iterons found at the origin of pPS10 replicon. These are brought together in parallel register by zipping-up DNA-bound RepA monomers. We also present evidence for a distinct role of RepA dimers in DNA looping. This work defines a new regulatory interface in Rep proteins.
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Early events in the binding of the pPS10 replication protein RepA to single Iteron and operator DNA sequences.
Journal of molecular biology, 2006Co-Authors: Teresa Díaz-lópez, Cristina Lucía Dávila-fajardo, Franca Blaesing, M. Pilar Lillo, Rafael GiraldoAbstract:RepA protein, encoded in the Pseudomonas pPS10 replicon, is a stable dimer in solution (dRepA), acting as a self-repressor of repA transcription through binding to an inverted repeat operator. However, RepA monomers (mRepA) are required to initiate plasmid replication upon binding to four directly repeated DNA sequences (Iterons). RepA is composed of two winged-helix (WH) domains: C-terminal WH2 is the main DNA-binding domain (DBD) for both target sequences, whereas N-terminal WH1 acts as dimerization interface in dRepA, but becomes a second DBD in mRepA. On the basis of CD spectroscopy, hydrodynamics, X-ray crystallography and model building studies, we proposed previously that the activation of RepA initiator implies a large structural change in WH1, coupled to protein monomerization and interdomain compaction. Here, we report novel features in the process. Binding curves of RepA to an Iteron, followed by fluorescence anisotropy in solution and by surface plasmon resonance on immobilized DNA, exhibit the profiles characteristic of transitions between three states. In contrast, RepA-R93C, a monomeric activated mutant, exhibits a single binding transition. This suggests the presence of an intermediate species in the Iteron-induced dissociation and structural transformation of RepA. High concentrations of bovine serum albumin or ovalbumin (macromolecular crowding) enhance RepA affinity for an Iteron in solution and, in gel mobility-shift assays, result in the visualization of novel protein-DNA complexes. RepA-induced DNA bending requires the binding of two WH domains: either both WH2 in dimers (operator) or WH1 plus WH2 in monomers (Iteron).
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Twenty years of the pPS10 replicon: insights on the molecular mechanism for the activation of DNA replication in Iteron-containing bacterial plasmids.
Plasmid, 2004Co-Authors: Rafael Giraldo, M. Elena Fernández-tresguerresAbstract:Abstract This review focuses on the contributions of the Pseudomonas replicon pPS10 to understanding the initiation of DNA replication in Iteron-containing plasmids from Gram-negative bacteria. Dimers of the pPS10 initiator protein (RepA) repress repA transcription by binding to the two halves of an inverted repeat operator. RepA monomers are the active initiator species that bind to four directly repeated sequences (Iterons). pPS10 initiator was the first Rep protein whose domains were defined (two “winged-helix,” WH modules) and their binding sites were identified at each half of the Iteron repeat. This was confirmed by the crystal structure of the monomer of a homologous initiator (RepE from F plasmid) bound to Iteron DNA. The recently solved structure of the dimeric N-terminal domain (WH1) of pPS10 RepA, when compared to the RepE monomer, shows that upon dimer dissociation an α-helix at WH1 C-terminus becomes part of an interdomain β-sheet. In solution, the Iteron sequence, by itself, can induce the same kind of structural transformation in RepA. This seems to alter the package of both WH domains to adapt their DNA reading heads (HTH motifs) to the distinct spacing between half repeats in Iterons and operator. Based on biochemical and spectroscopic work, structural and functional similarities were proposed between RepA and archaeal/eukaryal initiators. This was independently confirmed by the crystal structure of the archaeal initiator Cdc6. Characterization of mutants, either in pPS10 or in the Escherichia coli chromosome, has provided some evidence on a WH1-mediated interaction between RepA and the chromosomal initiator DnaA that results in a broadened-host range.
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structural changes in repa a plasmid replication initiator upon binding to origin dna
Journal of Biological Chemistry, 2003Co-Authors: Teresa Diazlopez, Marta Lagesgonzalo, Ana Serranolopez, Carlos Alfonso, German Rivas, Ramon Diazorejas, Rafael GiraldoAbstract:Abstract RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence inrepA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (Iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole Iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to Iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.
