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Results for “Replication initiation protein”

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At least 127 records · Page 7Linked to original sources

PubMed1990

N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication.

The replication initiation protein pi of the Escherichia coli plasmid R6K is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication. While the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region. By deleting C-terminal segments of the pi coding region, we have found that the N-terminal polypeptides of pi that are produced, corresponding to the first 117 and 164 amino acids, respectively, retain the negative activity of the bifunctional protein, i.e. these truncated pi proteins specifically inhibit R6K replication in vivo. These negatively acting polypeptides, however, are incapable of initiating replication in vivo and fail to bind to the gamma-origin of the R6K DNA in vitro. A correspondence between the observed negative activity of the N-terminal peptide and the negative regulatory activity of the intact pi protein is supported by the finding that point mutations introduced into the 164 amino acid N-terminal peptide that result in a decrease in its inhibitory activity also produce a plasmid high-copy phenotype when these mutations are incorporated into the full-length pi protein. These findings demonstrate that the negative domain of pi resides in the N-terminal segment of the protein. Furthermore, the data obtained suggest that inhibition of R6K replication by pi does not require direct binding to DNA.

Amino Acid Sequence↗

PubMed2005

Apoptosis in budding yeast caused by defects in initiation of DNA replication.

Apoptosis in metazoans is often accompanied by the destruction of DNA replication initiation proteins, inactivation of checkpoints and activation of cyclin-dependent kinases, which are inhibited by checkpoints that directly or indirectly require initiation proteins. Here we show that, in the budding yeast Saccharomyces cerevisiae, mutations in initiation proteins that attenuate both the initiation of DNA replication and checkpoints also induce features of apoptosis similar to those observed in metazoans. The apoptosis-like phenotype of initiation mutants includes the production of reactive oxygen species (ROS) and activation of the budding-yeast metacaspase Yca1p. In contrast to a recent report that activation of Yca1p only occurs in lysed cells and does not contribute to cell death, we found that, in at least one initiation mutant, Yca1p activation occurs at an early stage of cell death (before cell lysis) and contributes to the lethal effects of the mutation harbored by this strain. Apoptosis in initiation mutants is probably caused by DNA damage associated with the combined effects of insufficient DNA replication forks to completely replicate the genome and defective checkpoints that depend on initiation proteins and/or replication forks to restrain subsequent cell-cycle events until DNA replication is complete. A similar mechanism might underlie the proapoptotic effects associated with the destruction of initiation and checkpoint proteins during apoptosis in mammals, as well as genome instability in initiation mutants of budding yeast.

Apoptosis↗

PubMed2004

Eukaryotic MCM proteins: beyond replication initiation.

The minichromosome maintenance (or MCM) protein family is composed of six related proteins that are conserved in all eukaryotes. They were first identified by genetic screens in yeast and subsequently analyzed in other experimental systems using molecular and biochemical methods. Early data led to the identification of MCMs as central players in the initiation of DNA replication. More recent studies have shown that MCM proteins also function in replication elongation, probably as a DNA helicase. This is consistent with structural analysis showing that the proteins interact together in a heterohexameric ring. However, MCMs are strikingly abundant and far exceed the stoichiometry of replication origins; they are widely distributed on unreplicated chromatin. Analysis of mcm mutant phenotypes and interactions with other factors have now implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure. These experiments indicate that the MCMs are central players in many aspects of genome stability.

Animals↗

PubMed2004

Nanoviruses: genome organisation and protein function.

Nanoviruses, single-stranded DNA (ssDNA) plant viruses with a multipartite genome, share similarities with members of the Circoviridae family that infect mammals or birds as well as with the Geminiviridae, the only other plant virus family with circular ssDNA genomes. Although the virions of the latter are unique and different from that of the circoviruses, the mode of replication of viruses with monopartite or multipartite circular ssDNA genomes is strikingly similar. They multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyse initiation of ssDNA replication and resolution of replicative ssDNA into circular single-stranded virion DNA. All these ssDNA viruses exploit host polymerases for DNA synthesis and code for proteins that modulate the host's cell cycle favourably for virus multiplication. Recent three-dimensional structure analyses of a geminivirus and a parvovirus Rep protein have revealed an intriguing similarity between the catalytic domains of their respective Rep proteins. Furthermore, these structural data revealed that ssDNA virus replication initiator proteins might represent evolutionary intermediates between certain RNA-binding proteins and some multifunctional origin-binding proteins of papovaviruses.

Amino Acid Motifs↗

PubMed1997

Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?

pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter.

Bacterial Proteins↗

PubMed1994

Plasmids of the pT181 family show replication-specific initiator protein modification.

The rolling circle plasmids of Staphylococcus aureus regulate their replication by controlling initiator (Rep) protein synthesis. It was demonstrated recently that the pT181 initiator protein RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC* (A. Rasooly and R. P. Novick, Science, 262:1048-1050). We establish here that this initiator modification occurs with four other members of the pT181 family and that it occurs in Bacillus subtilis as well as S. aureus. These results suggest that Rep conversion to Rep* is probably universal among plasmids of the pT181 family and is not host dependent.

Amino Acid Sequence↗

PubMed2006

Role of ATP hydrolysis in the DNA translocase activity of the bovine papillomavirus (BPV-1) E1 helicase.

The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal approximately 300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects approximately 30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non-hydrolysable ATP analogue AMP-PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T(1/2) > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T(1/2) approximately 60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein-protein interactions switch from low to high affinity. This reciprocal change in protein-protein and protein-DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds.

Adenine Nucleotides↗

PubMed1988

Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli.

ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC). ADP bound to that site renders dnaA protein inactive for replication. Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein. The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E. coli phospholipid, is inactive. Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes. With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated. Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication. Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect. The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.

Adenosine Diphosphate↗

PubMed1996

The initiator protein E1 binds to the bovine papillomavirus origin of replication as a trimeric ring-like structure.

The replication initiator protein E1 binds to the origin of replication of bovine papillomavirus in several forms. E1 can bind to its recognition sequence as a monomer together with the viral transcription factor E2, or as a trimeric E1 complex. The trimerization of E1 is mediated by the sequence-specific binding of E1 to DNA, and results in an E1 complex that is linked topologically to the DNA because the three molecules of E1 form a ring-like structure that encircles the DNA. These results demonstrate that E1 utilizes unusual mechanisms for sequence-specific binding to DNA and for the generation of a structure that encircles the DNA. We believe that these forms of E1 bound to the origin of replication represent intermediates in a transition in the function of E1, from a sequence-specific origin of replication recognition protein to a form of E1 that is competent for the initiation of viral DNA replication.

Base Sequence↗

PubMed1995

Geminivirus replication: genetic and biochemical characterization of Rep protein function, a review.

Replication of the single-stranded DNA genome of plant geminiviruses follows a rolling circle mechanism. It strictly depends on a 'replication initiator protein' (Rep) which is the sole viral protein essential for replication. Rep protein catalyzes multiple reactions during the reproductive cycle of the virus. Here we summarize the recent advances of in vivo and in vitro analyses of the various Rep protein activities in a model for replication initiation and termination. In addition, the position of the geminivirus Rep protein within a general context of bacterial and mammalian replication initiator proteins is discussed.

Amino Acid Sequence↗

PubMed1986

Static and initiator protein-enhanced bending of DNA at a replication origin.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, DNA packaging, and the recognition of specific DNA sequences by proteins. It has recently been demonstrated that DNA bending can be sequence-directed. Bent DNA has also been observed as a consequence of sequence-specific binding of proteins to DNA. In this report DNA of plasmid pT181 is shown to contain a bend at the replication origin. Furthermore, this bend is enhanced by the binding of the pT181 replication initiator protein, RepC, to the origin.

Bacterial Proteins↗

PubMed2003

Structural insights into single-stranded DNA binding and cleavage by F factor TraI.

Conjugative plasmid transfer between bacteria disseminates antibiotic resistance and diversifies prokaryotic genomes. Relaxases, proteins essential for conjugation, cleave one plasmid strand sequence specifically prior to transfer. Cleavage occurs through a Mg(2+)-dependent transesterification involving a tyrosyl hydroxyl and a DNA phosphate. The structure of the F plasmid TraI relaxase domain, described here, is a five-strand beta sheet flanked by alpha helices. The protein resembles replication initiator protein AAV-5 Rep but is circularly permuted, yielding a different topology. The beta sheet forms a binding cleft lined with neutral, nonaromatic residues, unlike most single-stranded DNA binding proteins which use aromatic and charged residues. The cleft contains depressions, suggesting base recognition occurs in a knob-into-hole fashion. Unlike most nucleases, three histidines but no acidic residues coordinate a Mg(2+) located near the catalytic tyrosine. The full positive charge on the Mg(2+) and the architecture of the active site suggest multiple roles for Mg(2+) in DNA cleavage.

Binding Sites↗

PubMed1984

The bacteriophage lambda O and P protein initiators promote the replication of single-stranded DNA.

A soluble enzyme system that specifically initiates lambda dv plasmid DNA replication at a bacteriophage lambda replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176-6180] is also capable of replicating the single-stranded circular chromosomes of phages M13 and phi X174 to a duplex form. This chain initiation on single-stranded templates is novel in that it is absolutely dependent on the lambda O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the lambda chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein-dependent pre-priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single-stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage lambda in vivo.

Bacteriophage lambda↗

PubMed1995

Specific nicking-closing activity of the initiator of replication protein RepB of plasmid pMV158 on supercoiled or single-stranded DNA.

Asymmetric rolling circle replication of the promiscuous replicon pMV158 is initiated by the plasmid-encoded RepB protein. In vitro, purified RepB protein introduces a nick within the leading strand origin of replication by a nucleophylic attack on the phosphodiester bond at the dinucleotide GpA. Some changes within and around this dinucleotide were recognized by the protein. RepB nicked and closed supercoiled pMV158 DNA, having an optimum activity at 60 degrees C. We have imitated, in vitro, a process of rolling circle replication, since RepB was able to nick (initiation) and to covalently close (termination) single-stranded oligonucleotides containing the protein cleavage sequence. Covalent DNA-protein complexes were not found, indicating that RepB has unique features among plasmid-encoded proteins involved in rolling-circle replication or conjugative mobilization.

Bacterial Proteins↗

PubMed2001

Two compound replication origins in Saccharomyces cerevisiae contain redundant origin recognition complex binding sites.

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

Base Sequence↗

PubMed1990

Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro.

The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained. We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective. Augmentation of the activity of a limiting level of the ATP form of dnaA protein by the otherwise inert ADP form implies that at some stage of initiation both forms are active. The dnaA protein is essential up to the stage of forming the prepriming complex; upon salt dissociation from an oriC complex, the protein can be recycled to function at a fresh origin. Distinctive conformational states of the ATP form are implied by interactions with oriC DNA, by the influence of phospholipids on accelerating nucleotide exchange, and by the susceptibility to proteolytic cleavage.

Adenosine Diphosphate↗

PubMed1996

Complete nucleotide sequence of the Sulfolobus islandicus multicopy plasmid pRN1.

The complete sequence of the 5350-bp plasmid pRN1 from the crenarchaeote Sulfolobus islandicus has been determined. This plasmid is the first to be sequenced from this group of thermoacidophilic archaebacteria (Archaea) and its high copy number and wide host range make it a good candidate for a cloning vector. pRN1 contains several open reading frames, including one that spans over half the plasmid and has significant similarity to the helicase domain of viral primase proteins. Directly upstream of this putative primase is a homologue of Cop, a family of small proteins from promiscuous eubacterial plasmids which control copy number by repressing the expression of the replication initiation protein. In eubacterial plasmids cop is found upstream of the replication initiator protein. The location of a cop homologue upstream of a primase-like gene in pRN1 suggests that it controls DNA replication in a manner similar to these eubacterial plasmids, but does so using a mixture of components from plasmids and viruses.

Amino Acid Sequence↗

PubMed1987

ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome.

ATP is bound to dnaA protein with high affinity (KD = 0.03 microM) and hydrolyzed slowly to ADP in the presence of DNA. ADP is also bound tightly to dnaA protein and exchanges with ATP very slowly. The ATP form is active in replication; the ADP form is not. A unique conformation of oriC, formed in an early initiation stage, depends on dnaA protein being in the ATP form. The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP. Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATP gamma S can replace ATP, the ATP function appears to be allosteric. The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle.

Adenosine Triphosphate↗