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Results for “Replication initiation protein”

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PubMed1983

The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding.

We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E. coli. This fusion was carried out with recombinant DNA methods. The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion. Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome. The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.

Bacterial Proteins↗

PubMed1999

Plasmid replication initiator protein RepD increases the processivity of PcrA DNA helicase.

The replication initiator protein RepD encoded by the Staphylococcus chloramphenicol resistance plasmid pC221 stimulates the helicase activity of the Bacillus stearothermophilus PcrA DNA helicase in vitro. This stimulatory effect seems to be specific for PcrA and differs from the stimulatory effect of the Escherichia coli ribosomal protein L3. Whereas L3 stimulates the PcrA helicase activity by promoting co-operative PcrA binding onto its DNA substrate, RepD stimulates the PcrA helicase activity by increasing the processivity of the enzyme and enables PcrA to displace DNA from a nicked substrate. The implication of these results is that PcrA is the helicase recruited into the replisome by RepD during rolling circle replication of plasmids of the pT181 family.

Adenosine Triphosphatases↗

PubMed2003

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin.

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.

Amino Acid Sequence↗

PubMed1997

Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA.

The bacteriophage lambda P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNA-binding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P x DnaB or a DnaB x DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the lambda O replication initiator enhances interaction of the P x DnaB complex with ssDNA. Partial disassembly of a ssDNA:O x P x DnaB complex by the DnaK/DnaJ/GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.

Bacterial Proteins↗

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↗

PubMed2003

Replication-initiator protein (UL9) of the herpes simplex virus 1 binds NFB42 and is degraded via the ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway plays a critical role in the degradation of short-lived and regulatory proteins in a variety of cellular processes. The F-box proteins are part of the ubiquitin-ligase complexes, which mediate ubiquitination and proteasome-dependent degradation of phosphorylated proteins. We previously identified NFB42, an F-box protein that is highly enriched in the nervous system, as a binding partner for the herpes simplex virus 1 UL9 protein, the viral replication-initiator protein, in a yeast two-hybrid screen. In the present work, we find that coexpression of NFB42 and UL9 genes in 293T cells leads to a significant decrease in the level of UL9 protein. Treatment with the 26S-proteasome inhibitor MG132 restores the UL9 protein to normal levels. We have observed also that the UL9 protein is polyubiquitinated in vivo and that the interaction between NFB42 and the UL9 protein is dependent upon phosphorylation of the UL9 protein. These results suggest that the interaction of the UL9 protein with NFB42 results in its polyubiquitination and subsequent degradation by the 26S proteasome. They suggest further a mechanism by which latency of herpes simplex virus 1 can be established in neuronal cells.

Cell Cycle Proteins↗

PubMed2006

Analogous expression pattern of Plasmodium falciparum replication initiation proteins PfMCM4 and PfORC1 during the asexual and sexual stages of intraerythrocytic developmental cycle.

DNA replication takes place at five different stages during the life cycle of Plasmodium falciparum including the human and mosquito hosts. DNA replication initiation, the rate-determining step is poorly understood in Plasmodium. Here we show that PfMCM4 and PfORC1, two members of prereplication initiation complex are expressed specifically in the nucleus during the trophozoite and schizont stages of the asexual parasitic life cycle where maximum amount of DNA replication takes place. Further, we show that these proteins are also expressed in gametocytes, where DNA replication also occurs. These results expand our knowledge on these proteins and resolves discrepancies arising from previous studies with respect to the expression pattern of replication initiation proteins during the parasite's life cycle.

Animals↗

PubMed1995

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells.

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.

Base Sequence↗

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↗

PubMed1999

Crystallization and preliminary X-ray diffraction studies of a replication initiator protein (RepE54) of the mini-F plasmid complexed with iteron DNA.

A replication initiator protein (RepE54) complexed with iteron DNA at its binding site was crystallized by the hanging drop vapor diffusion method. The crystals belong to monoclinic space group C2 with unit cell dimensions of a = 108.4 A, b = 81.9 A, c = 73.9 A, and beta = 121.5 degrees, where one molecule of the protein-DNA complex exists per asymmetric unit. They diffract X-rays up to 2.6 A resolution with synchrotron radiation.

Bacterial Proteins↗

PubMed1995

The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number.

Plasmids control their copy number by limiting the amount of the initiator for DNA replication. The plasmid pUB110 initiator protein is termed RepU. Expression of the pUB110 repU gene is controlled by two antisense RNAs that interfere with repU mRNA translation. Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms. The repU gene product was radiolabeled and purified by monitoring the radioactive label. RepU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactivation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal sequence expected for the repU gene. The RepU/RepU* protein mixture bound specifically to dso. At low protein concentrations, about six RepU/RepU* protomers bound to the dso region. At higher concentrations, an extended nucleoprotein complex was formed. The promoter for the repU gene was localized downstream of the dso region. The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization. This provides an additional copy number control by limiting RepU concentration. Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication initiation.

Amino Acid Sequence↗

PubMed2000

Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75.

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topo-isomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.

Amino Acid Sequence↗

PubMed2000

Activities associated with the putative replication initiation protein of coconut foliar decay virus, a tentative member of the genus Nanovirus.

The putative replication initiation protein (Rep) of Coconut foliar decay virus (CFDV) was expressed as a 6x His recombinant protein in E. coli and in recombinant baculovirus. Purified 6x His-Rep protein was demonstrated to possess sequence non-specific RNA- and ssDNA-binding activities as well as magnesium-dependent ATPase/GTPase activity. The yeast two-hybrid system revealed that CFDV Rep could interact with itself. Subcellular distribution of the CFDV Rep was studied by fractionation of insect cells infected with recombinant baculovirus expressing the 6x His-Rep protein and by laser scanning confocal microscopy of Nicotiana benthamiana epidermal cells bombarded with a construct encoding CFDV Rep fused to GFP. It was shown that CFDV Rep associated predominantly with nuclei and membranes of infected/transfected cells. These activities of CFDV-encoded Rep are very similar to those reported for Reps of geminiviruses.

Adenosine Triphosphatases↗

PubMed1983

Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA.

Initiation of DNA replication in plasmid R6K is potentiated by the plasmid-encoded 35 kd replication initiator protein. We had previously reported that the initiator bound to two regions of R6K DNA called Site I and Site II. Using DNAase I footprinting technique we have demonstrated that the initiator bound to seven tandem repeats of a 22 bp long sequence in Site I. In Site II, the initiator bound to a single repeat having the same consensus sequence and to two partial repeats that most likely overlap the promoter of the initiation protein cistron. Using dimethyl sulfate as a chemical probe, we have determined the purine residues of Site I and Site II that make contact with the initiator protein. The results show that eight out of nine contact points per repeat in Site I were located on one of the two strands of the DNA. The binding of the initiator to the Site II sequence could explain the observed autoregulation of the synthesis of the initiator protein by promoter occlusion.

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↗

PubMed2003

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA.

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli

Bacterial Proteins↗

PubMed2000

Identification of a potential membrane-targeting region of the replication initiator protein (TrfA) of broad-host-range plasmid RK2.

Plasmid RK2 codes for two species of the replication initiator protein TrfA (33 and 44 kDa). Both polypeptides are strongly associated with membrane fractions of Escherichia coli host cells (W. Firshein and P. Kim, Mol. Microbiol. 23, 1-10, 1997). We investigated the role of a 12-amino-acid hydrophobic region (HR) in the membrane association of TrfA. Epitope-tagged polypeptide fragments of TrfA that contained HR were expressed and found to be associated with membrane fractions. Site-directed mutagenesis of trfA revealed that changes of specific amino acids in HR can affect both TrfA association with the membrane and its ability to support replication of an RK2 oriV plasmid in vivo. These results are consistent with the hypothesis that membrane association of TrfA is functionally relevant and that the HR region of TrfA is involved in membrane association and DNA replication in vivo.

Bacterial Proteins↗

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↗