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Results for “Replication initiation protein”

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At least 19 recordsLinked to original sources

PubMed1992

Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins.

It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family. Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA. These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication. It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick. The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain. By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately. The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed.

Amino Acid Sequence↗

PubMed1991

Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101.

We have investigated the interaction of the host-encoded DNA bending protein IHF, the host-encoded initiator DnaA, and the plasmid-encoded initiator RepA with the replication origin of pSC101. We have discovered that DNA bending induced by IHF in vitro promoted the interaction of DnaA protein with two physically separated binding sites called dnaAs and dnaAw. This cooperative interaction at a distance, most probably, caused looping out of the ihf site. We have also discovered that RepA protein binding to its cognate sites promoted enhanced binding of DnaA protein to the physically distant dnaAs site, probably also by DNA looping. The addition of RepA to a binding reaction containing IHF and DnaA further enhanced the binding of DnaA protein to the dnaAs site. Thus, the three DNA-binding proteins interacted with the origin, generating a higher order structure in vitro. On the basis of the results of the known requirement of all three proteins for replication initiation, we have proposed a model for the structure of a preinitiation complex at the replication origin.

Bacterial Proteins↗

PubMed1991

Exonuclease III promotes in vitro binding of the replication initiator protein of plasmid pSC101 to the repeated sequences in the ori region.

Purified Rep protein, a replication initiator protein of plasmid pSC101, has less binding affinity for the direct repeats (DR) in the replication origin region (ori) than that for the inverted repeats (IR) in the promoter region of the structure gene of Rep (rep) (Sugiura, S. et al. (1990) J. Biochem. 107, 369-376). We found a protein factor that promotes binding of purified Rep to the DR sequence in the cell extract of Escherichia coli. In the presence of the factor, DNA fragments containing the DR sequence can form a specific DNA-protein complex by the addition of low concentrations of Rep. On the contrary, IR-containing DNA loses its binding activity for Rep by preincubation with the factor. We purified extensively the factor and identified it as exonuclease III (exo III). Enzymatic action of the factor or authentic exo III at 37 degrees C is necessary for binding of Rep to DR-DNA. This binding of Rep to duplex DNA treated with exo III is DR-sequence specific. Since Rep cannot bind to the single stranded DR sequence, the present finding suggests that partial single-stranded regions around the DR sequence are required for binding of Rep.

Bacterial Proteins↗

PubMed1989

Double-strand cleavage and strand joining by the replication initiator protein of filamentous phage f1.

The replication initiator protein (gene II protein (gpII] of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA. The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication. Upon completion of a round of synthesis, gpII cleaves and circulaizes the displaced single strand. When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in the presence of Mg2+, does not require superhelicity. The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand. The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg2+. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a onebase 3'-overhang. This reaction probably reflects the cleavage-joining activity of gpII in the termination event.

Base Sequence↗

PubMed1986

Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication.

The replication of the pT181 plasmid is dependent on the plasmid-encoded initiator protein RepC. We have previously shown that RepC protein has sequence-specific endonuclease and topoisomerase-like activities. In this paper we demonstrate that this initiator protein has sequence-specific DNA-binding properties. Based on filter binding of plasmid restriction fragments, RepC protein specifically recognizes only the pT181 origin region. Using DNase I and neocarzinostatin "footprinting" techniques, we show that RepC protein specifically binds to a 32-base-pair sequence within the origin that is part of the initiator cistron. Using dimethyl sulfate as a chemical probe, we have identified the purine residues that interact with the initiator protein. The features of the DNA region that interacts with RepC protein include sequences with the potential to form Z DNA and/or hairpin structures. The specific DNA-protein interaction at the origin may be critical in the initiation of pT181 DNA replication by RepC protein in association with other host initiation proteins.

Bacterial Proteins↗

PubMed1991

Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA.

The trfA gene, encoding the essential replication initiation protein of the broad-host-range plasmid RK2, possesses an in-frame overlapping arrangement. This results in the production of TrfA proteins of 33 and 44 kDa, respectively. Utilizing deletion and site-specific mutagenesis to alter the trfA operon, we compared the replication of an RK2-origin plasmid in several distantly related gram-negative bacteria when supported by both TrfA-44 and TrfA-33, TrfA-33 alone, or TrfA-44/98L (a mutant form of the TrfA-44 protein) alone. TrfA-44/98L is identical to wild-type TrfA-44 with the exception of a single conservative amino acid alteration from methionine to leucine at codon 98; this alteration removes the translational start codon for the TrfA-33 protein. Copy number and stability were virtually identical for plasmids containing both TrfA-44 and TrfA-33 proteins or TrfA-44/98L alone in Pseudomonas aeruginosa and Agrobacterium tumefaciens, two unrelated bacteria in which TrfA-33 is poorly functional. This, along with recent in vitro studies comparing TrfA-44, TrfA-33, and TrfA-44/98L, suggests that the functional activity of TrfA-44 is not significantly affected by the 98L mutation. Analysis of minimal RK2 derivatives in certain gram-negative bacterial hosts suggests a role of the overlapping arrangement of trfA in facilitating the broad host range of RK2. RK2 derivatives encoding TrfA-44/98L alone demonstrated decreased copy number and stability in Escherichia coli and Azotobacter vinelandii when compared with derivatives specifying both TrfA-44 and TrfA-33. A strategy employing the trfA-44/98L mutant gene and in vivo homologous recombination was used to eliminate the internal translational start codon of trfA in the intact RK2 plasmid. The mutant intact RK2 plasmid produced only TrfA-44/98L. A small reduction in copy number and beta-lactamase expression resulted in E. coli, suggesting that overlapping trfA genes also enhance the efficiency of replication of the intact RK2 plasmid.

Amino Acid Sequence↗

PubMed1985

The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron.

The plasmid-encoded replication initiator protein of pSC101 specifically repressed initiation of transcription of its own cistron from its natural promoter. Addition of the purified initiator had little or no visible effect on transcription initiated from a heterologous promoter. DNase protection experiments revealed that the RNA polymerase recognition sequence was overlapped by the initiator protein recognition sequences, which are vicinal to the replication origin. Using the labeled promoter sequence, we have performed competitive DNase protection experiments in two ways: by adding RNA polymerase and initiator protein simultaneously or by sequentially adding first RNA polymerase and then initiator protein to the DNase reaction mixture. The RNA polymerase protection pattern was recessive to that of the initiator regardless of whether the two proteins were added simultaneously or sequentially. This observation suggests that the mechanism of autoregulation is due to competition of the two proteins for the sequences in and around the promoter region. Furthermore, the sequential addition experiments raise the possibility of displacement of RNA polymerase from the promoter by the initiator protein.

Bacterial Proteins↗

PubMed1990

DNA binding properties of purified replication initiator protein (Rep) encoded by plasmid pSC101.

We have purified the replication initiator protein (Rep) coded by plasmid pSC101. The purified protein was confirmed to be Rep by its amino-terminal sequence. Rep exists as a dimer and has a sequence-specific DNA-binding property. Protection experiments with DNA against cleavage by DNase I or exonuclease III showed that Rep bound preferentially to two nearly dyad-symmetric sequences overlapping the promoter of the rep gene, a structure gene of Rep. Transcripts in vitro from the rep promoter were identified and the precise initiation sites were determined by the primer-extension method. Rep represses the transcription from the rep promoter but not that from the bla gene promoter in the same reaction mixture, that is the rep gene is autoregulated. The replication origin (ori) of the plasmid contains directly repeated sequences similar to the symmetric sequences. However, a one order of magnitude higher concentration of the protein is required to bind to the origin repeats.

Amino Acid Sequence↗

PubMed1992

Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi.

The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA. In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected. We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough. Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation. Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene. The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo. We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight. Thus, at least three options exist in the translation of the wt pir mRNA. Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38. Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).

Bacterial Proteins↗

PubMed1985

Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step.

The replication initiator protein of plasmid R6K preferentially repressed transcription initiated in vitro from the promoter of the initiator protein cistron. DNase I protection experiments revealed that the sequences in the region of the promoter recognized by the initiator protein partially overlapped the sequences of the same promoter recognized by RNA polymerase of Escherichia coli. Competitive DNase I protection experiments revealed that the initiator not only prevented the RNA polymerase from binding to the promoter sequence but also displaced RNA polymerase from preformed enzyme-promoter binary complexes. Thus, the initiator protein acts as a transcriptional repressor of its own cistron by either preventing RNA polymerase from binding to the promoter or by displacing RNA polymerase from promoter-enzyme complexes.

Bacterial Proteins↗

PubMed1992

A single amino acid substitution reduces the superhelicity requirement of a replication initiator protein.

The origin of rolling circle replication in filamentous coliphage consists of a core origin that is absolutely required and an adjacent replication enhancer sequence that increases in vivo replication 30 to 100-fold. The core origin binds the initiator protein (gpII) which either nicks or relaxes negatively superhelical replicative form DNA (RFI). Nicking at the origin, but not relaxation, leads to initiation of DNA replication. Our results indicate that the ratio of nicking to relaxation (nicking-closing) in vitro depends on the superhelical density of the substrate. We have studied the effect of a single amino acid substitution in gpII, which allows wild-type levels of replication in the absence of the enhancer, on origin nicking and binding. The enhancer-independent mutation yields more nicking and less relaxation of RFI, compared to the wild-type protein. The mutant gpII also shows a reduced requirement for superhelicity of the substrate in the nicking reaction. At the same time, the mutant gpII increases the cooperativity of protein-protein interactions in origin binding. We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both increase in the negative superhelicity of the substrate and action of the replication enhancer may antagonize the relaxation activity.

Amino Acid Sequence↗

PubMed1991

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.

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.

Bacterial Proteins↗

PubMed1992

Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.

We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.

Amino Acid Sequence↗

PubMed1991

The effect of plasmid copy number mutations on pT181 replication initiator protein expression.

Previous studies have shown that plasmid pT181 controls its replication by countertranscript-mediated regulation of the rate of synthesis of the pT181 initiator, RepC. In this study, the relation has been studied between plasmid copy number and RepC synthesis for a series of pT181 copy number mutants. For each mutant plasmid, the repC coding sequence along with its 5' regulatory region was translationally fused to the beta-lactamase structural gene on a vector plasmid unrelated to pT181. By means of these constructs, the effect of regulatory mutations on the initiator synthesis could be measured at constant copy number. With one exception, the mutant control regions showed elevated beta-lactamase activity in comparison to the wild-type. However, the relative increase was not very well correlated with the copy number of the corresponding mutant plasmid. The possibility is considered that factors such as DNA secondary structure may have important ancillary effects on the regulation mechanism.

Bacterial Proteins↗

PubMed1988

In-vivo studies on the cis-acting replication initiator protein of IncFII plasmid NR1.

Using segment-directed mutagenesis, a temperature-sensitive mutant of the gene that encodes the cis-acting RepA1 initiation protein of the IncFII plasmid NR1 was isolated. The mutant protein was unable to promote initiation of plasmid replication in vivo at 42 degrees C. Both the wild-type and the mutant repA1 genes were cloned separately into the high-expression vector plasmid pAS1. In these pAS1-repA1 derivatives, the transcription of the repA1 gene was under the control of the lambda PL promoter, which was regulated by the temperature-sensitive lambda cI857 repressor protein. The translation initiation of the repA1 mRNA from these derivatives was mediated by the lambda cII Shine-Dalgarno sequence and initiation codon. The yield of 33,000 Mr RepA1 protein detected on SDS/polyacrylamide gels from Escherichia coli cells containing the pAS1-repA1 derivatives was dependent upon whether the newly synthesized RepA1 was capable of interacting in cis with the downstream NR1 replication origin on the cloned DNA fragment. Mutations in the repA1 gene or deletions of the cis origin region dramatically increased the detectable yield of RepA1 protein. Deletion of the NR1 origin region from the pAS1 derivative containing the wild-type repA1 gene enabled the cis-acting RepA1 protein to complement partially the temperature-sensitive repA1 mutant in trans, to increase the copy number in trans of plasmids that contained the NR1 replicon, and to help NR1 derivatives overcome plasmid incompatibility. The trans effects of RepA1 provided by the pAS1-repA1 derivatives that retained the origin in cis were much less significant. RepA1 provided in trans also stimulated the replication of plasmids carrying cloned copies of the NR1 replication origin region regardless of whether the origin was transcribed from an upstream promoter.

Amino Acid Sequence↗

PubMed1985

The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities.

Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein. Here we demonstrate that highly purified RepC protein has sequence-specific endonuclease and topoisomerase-like activities. A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking-closing by RepC protein. RepC introduces a single strand break within the pT181 origin. The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence. This nick site probably corresponds to the start site of pT181 replication. The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism.

Bacterial Proteins↗

PubMed1987

A replication initiator protein enhances the rate of hybrid formation between a silencer RNA and an activator RNA.

The replication origin gamma of plasmid R6K in certain miniplasmids is kept silent by a silencer RNA. We have identified a major and three minor transcripts that are synthesized in a direction antiparallel and complementary to the silencer RNA. The major RNA, called the activator, is essential for replication from ori gamma. The complementary nature of the activator and silencer RNAs strongly suggests that the former is a target of the latter. We have also discovered that the initiator protein is a sequence-specific double-stranded RNA-binding protein that accelerates the rate of activator-silencer hybrid formation. Thus the efficient silencing of ori gamma can be explained by silencer RNA-activator RNA hybrid formation that is driven to completion by the initiator protein.

DNA Replication↗

PubMed1988

A novel role for cAMP in the control of the activity of the E. coli chromosome replication initiator protein, DnaA.

DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.

Amino Acid Sequence↗