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Results for “Replication initiation protein”

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PubMed2001

Regulation of premeiotic S phase and recombination-related double-strand DNA breaks during meiosis in fission yeast.

The meiotic cell cycle is characterized by high levels of recombination induced by DNA double-strand breaks (DSBs), which appear after completion of premeiotic S phase, leading to the view that initiation of recombination depends on meiotic DNA replication. It has also been indicated that DNA replication initiation proteins may differ between the meiotic and mitotic cell cycles, giving rise to an altered S phase, which could contribute to the high level of recombination during meiosis. We have investigated these possibilities in the fission yeast Schizosaccharomyces pombe and found that core DNA replication initiation proteins used during the mitotic cell cycle, including Cdc18p (budding yeast Cdc6p), Cdc19p (Mcm2p), Cdc21p (Mcm4p) and Orp1p (Orc1p), are also required for premeiotic S phase. Reduced activity of these proteins prevents completion of DNA replication but not formation of DSBs. We conclude that recombination-related DSB formation does not depend on the completion of meiotic DNA replication and we propose two parallel developmental sequences during the meiotic cell cycle: one for premeiotic S phase and the other for initiating recombination.

Cell Cycle Proteins↗

PubMed1991

A single-stranded region located downstream of the repeated sequence in the ori region of pSC101 is required for binding of the Rep protein in vitro.

Purified replication initiator protein (Rep) of plasmid pSC101 binds preferentially to two inverted repeats (IR) overlapping the promoter of its own structure gene, rep. However, the protein has much lower binding affinity for directly repeated (DR) sequences in the replication origin (ori) that are similar to the symmetric sequences. Exonuclease III (exo III) promotes in vitro binding of Rep to the origin repeats. In the present studies, DNA containing the DR sequences was degraded unidirectionally by exo III and then formed a complex with Rep. Analyses of DNA from the complex revealed that Rep bound to the DR sequences only when the degradation proceeded from the 3' end proximal to IR to the DR sequences, resulting in conversion of the duplex structure in a specific downstream region of DR into the single-stranded form. The degradation in the opposite direction had no effect on binding of Rep. These results suggest that a localized structural change of DNA adjacent to DR is required for Rep binding to double-stranded DR sequences. By contrast, exo III strikingly inhibited binding of Rep to DNA containing the IR sequences by introducing a single-stranded moiety into duplex IR sequences.

Adenosine Triphosphatases↗

PubMed1994

Expression of Caulobacter dnaA as a function of the cell cycle.

The initiation of DNA replication is under differential control in Caulobacter crescentus. Following cell division, only the chromosome in the progeny stalked cell is able to initiate DNA replication, while the chromosome in the progeny swarmer cell does not replicate until later in the cell cycle. We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C. crescentus. Analysis of the cloned C. crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli. The gene is located 2 kb from the origin of replication. Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter. Immunoprecipitation of a DnaA'-beta-lactamase fusion protein showed that although expression occurs throughout the cell cycle, there is a doubling in the rate of expression just prior to the initiation of replication.

Amino Acid Sequence↗

PubMed1997

The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences.

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.

Amino Acid Sequence↗

PubMed1996

A leucine zipper motif determines different functions in a DNA replication protein.

RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors.

Amino Acid Sequence↗

PubMed2004

Staphylococcus aureus multiresistance plasmid pSK41: analysis of the replication region, initiator protein binding and antisense RNA regulation.

The vast majority of large staphylococcal plasmids characterized to date appear to possess an evolutionarily common replication system, which has clearly had a major impact on the evolution of antimicrobial resistant staphylococci worldwide. Related systems have also been found in plasmids from other Gram-positive genera, including enterococci, streptococci and bacilli. The 46.4 kb plasmid pSK41 is the prototype of a family of conjugative staphylococcal multiresistance plasmids. The replication region of pSK41 encodes a protein product, Rep, which was shown to be essential for replication; mutations that truncated Rep could be complemented in trans. Rep was found to bind in vitro to four tandem repeat sequences located centrally within the rep coding region. An A + T-rich inverted repeat sequence upstream of rep was required for efficient replication, whereas no sequences downstream of rep were necessary. An antisense countertranscript, RNAI, encoded upstream of rep was identified and transcriptional start points for both RNAI and the rep-mRNA were defined.

Amino Acid Sequence↗

PubMed1987

P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.

Cloning, Molecular↗

PubMed1995

Replication enhancer requirement for recognition of heterologous replication origin by an initiator protein.

In the pT181 plasmid family, the replication initiation protein (Rep) encoded by each plasmid recognizes only its cognate origin, unless the Rep protein is expressed at abnormally high levels. Heterologous recognition of the origin of the pC221 plasmid by the RepC protein of the pT181 plasmid requires that cmp, the pT181 replication enhancer, be present on the same plasmid as the origin of replication. These findings indicate that cmp has a role in the specificity of Rep-ori recognition and support the model that cmp facilitates the formation/stabilization of the RepC-origin complex.

Bacterial Proteins↗

PubMed1998

The human papillomavirus type 18 (HPV18) replication protein E1 is a transcriptional activator when interacting with HPV18 E2.

The human papillomavirus type 18 E1 and E2 proteins are both required for the initiation of viral DNA replication. Whereas E2 is the major viral transcription regulator, E1 is the replication initiator protein. They interact with each other and with the origin sequences to initiate viral DNA replication. We show that the HPV18 E1 and E2 proteins, when bound to an origin sequence cloned upstream of a heterologous promoter, synergistically activate transcription. This synergy required binding of E2 to at least two binding sites, but was partially independent of E1 binding to the origin of replication. Transcriptional activation was observed even in the absence of replication of the target DNA. Only homologous E1 and E2 proteins binding to homologous origin sequences from BPV1 or HPV18 viruses could synergistically activate transcription. We show that the HPV18 E1 protein can activate transcription when targeted to the DNA by fusion of the complete polypeptide with the BPV1 E2 C-terminus dimerization/DNA binding domain, implying that HPV18 E1 is an intrinsic transcriptional activator, though less potent than E2. The interaction between E1 and E2 may form a transcriptionally active complex during initiation of viral DNA replication.

Chloramphenicol O-Acetyltransferase↗

PubMed1994

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.

Amino Acid Sequence↗

PubMed2006

Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9.

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.

Amino Acid Sequence↗

PubMed1996

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.

Bacterial Proteins↗

PubMed2004

Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid.

The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5'-TCTTAA-3', which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

Bacterial Proteins↗

PubMed2004

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Adenosine Triphosphatases↗

PubMed1992

Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K.

A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.

Bacterial Proteins↗

PubMed1985

Binding and bending of the lambda replication origin by the phage O protein.

We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19-bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA-protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA-protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non-symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor-operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.

Alkylation↗

PubMed1998

Protein domains and conformational changes in the activation of RepA, a DNA replication initiator.

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

Amino Acid Sequence↗

PubMed1999

Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata.

The basic replicon of the narrow-host-range plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized. The replicon consists of a 1.7-kb DNA fragment, which contains the genetic information required for autonomous replication and stable maintenance. Analysis of its sequence revealed the presence of two ORFs which seem to form one transcription unit. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology to the theta-replicase (RepA) protein of ColE2 plasmid and to the RepA proteins of a family of replicons from gram-positive and gram-negative bacteria, also related to ColE2. The product encoded by ORF2 showed a certain similarity to the RepB proteins of the same family of replicons and perhaps represents the pHE1 RepB function. Deletion analysis suggests that the pHE1 origin of replication (ori) is located in an 800-bp region upstream of repA. A third putative gene, incA, was found on the complementary strand to the leader region of the repA mRNA. This, together with the presence in the 5' untranslated region of the repA mRNA of inverted repeats that could form stable stem-loop structures, suggests that the incA gene encodes a small antisense RNA. A possible control mechanism for pHE1 replication is proposed, involving an RNA molecule which sequesters the translational initiation region of the replication protein RepA. The basic replicon characterized here shows very interesting properties that should allow it to be used in the construction of cloning and expression vectors for moderate halophiles.

Amino Acid Sequence↗