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PubMed2004

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer.

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.

Bacterial Proteins↗
PubMed1997

Autoregulation of the Escherichia coli replication initiator protein, DnaA, is indirect.

The expression of dnaA is autoregulated, in that transcription of the gene increases when DnaA is inactivated (and initiation of replication prevented) and decreases when DnaA is supplied in excess. However, the inactivation of DnaA does not necessarily lead to increased DnaA production, as dnaA(Ts; temperature sensitive) strains which are integratively suppressed by derivatives of the plasmid R1 do not show temperature-induced derepression. Several possible explanations for this unanticipated behaviour were considered and ruled out. We suggest here that the completion of a critical step in initiation may prevent dnaA derepression: although DnaA would be required to complete this step at oriC, DnaA(Ts) would be sufficient at the R1 origin. Autoregulation of dnaA has been attributed to the binding of DnaA at a consensus binding site in the dnaA promoter region. We show here, using reporter systems, that this DnaA-binding site is not required for the autoregulatory response. We find, further, that replacement of the chromosomal dnaA gene with one containing a mutated binding site causes no demonstrable phenotypic change: cells with the mutant gene show no disadvantage in competition with dnaA+ cells.

Bacterial Proteins↗
PubMed2003

pHP489, a Helicobacter pylori small cryptic plasmid, harbors a novel gene coding for a replication initiation protein.

We have analyzed a Helicobacter pylori plasmid, pHP489. The 1222-bp nucleotide sequence had one open reading frame, a DnaA-binding site, one direct repeat, and three inverted repeats. The (G+C) content of pHP489 was 33.3%. Although the nucleic acid sequence and deduced amino acid sequence were homologous to those of other bacterial plasmid Rep proteins, the degree of similarity was very low. A deletion analysis showed that the Rep protein was not required for the replication of pHP489 in its H. pylori host, but the host replication machinery was needed.

Amino Acid Sequence↗
PubMed1997

Prediction of the structure of the replication initiator protein DnaA.

The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an "open twisted alpha/beta structure," containing five alpha-helices alternating with five beta-strands. In our proposed structural model the interior of the core is formed by a parallel beta-sheet, whereas the alpha-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first beta-strand. The N-terminal domain (80 aa) is composed of two alpha-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a beta-strand and an additional alpha-helix. The N-terminal domain and the alpha/beta core region of DnaA are connected by a variable loop (45-70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly alpha-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal alpha-helices could interact with each other, forming an internal "coiled coil" and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts.

Amino Acid Sequence↗
PubMed2002

Nuclear organization of DNA replication initiation proteins in mammalian cells.

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.

Cell Cycle Proteins↗
PubMed1992

Uncoupling of the DNA topoisomerase and replication activities of an initiator protein.

The replication initiator proteins encoded by the pT181 and related plasmids have sequence-specific DNA binding and topoisomerase activities. These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiation of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, and replication activities, we have carried out site-directed mutagenesis of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studies also showed that the DNA topoisomerase activity of the initiator protein can be uncoupled from its tight noncovalent DNA binding and replication activities.

Bacterial Proteins↗
PubMed1994

Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution.

The replication initiator protein pi of plasmid R6K binds seven 22 bp direct repeats (DR) in the gamma origin. The pi protein also binds to an inverted repeat (IR) in the operator of its own gene, pir, which lies outside the gamma origin sequences. A genetic system was devised to select for pi protein mutants which discriminate between IR and DR (York et al., Gene (Amst.) 116, 7-12, 1992; York and Filutowicz, J. Biol. Chem. 268, 21854-21861, 1993). From this selection the mutant pi S87N protein was isolated which is deficient in repressing the pir gene's expression because it cannot bind to IR at the pir gene operator. Remarkably, we discovered that pi S87N binds to DR cooperatively under conditions where wt pi binds independently. Moreover, the pi S87N is more active as a replication initiator in vivo when supplied at the same level as wt pi. Quantitative binding assays showed that both wt pi and pi S87N bind a DNA fragment containing a single DR unit with a similar affinity (Kd = 0.3 x 10(-12) M). Thus, cooperativity of pi S87N is most likely achieved through altered interactions between promoters bound at adjacent DR units.

Bacterial Proteins↗
PubMed1985

Conformational changes in a replication origin induced by an initiator protein.

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.

Bacterial Proteins↗
PubMed1993

Replication-specific inactivation of the pT181 plasmid initiator protein.

Replication of the Staphylococcus aureus plasmid pT181, which occurs by the rolling circle mechanism, is accompanied by the covalent attachment of a approximately 12-residue oligodeoxy-nucleotide to one subunit of the dimeric plasmid-coded initiator protein, RepC. This oligonucleotide represents the plasmid sequence immediately 3' to the initiating nick site. The resulting heterodimeric protein lacks the topoisomerase and replication activities of unmodified RepC, suggesting that the regulation of plasmid DNA replication requires post-replicational inactivation of the initiator protein as well as control of its synthesis.

Bacterial Proteins↗
PubMed2001

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Bacteriophage lambda↗
PubMed1999

Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101.

Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA-DnaB interaction cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo. Although, interaction of DnaA with DnaB could not substitute for RepA-DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.

Bacterial Proteins↗
PubMed2000

ATPase switches controlling DNA replication initiation.

Proteins that bind and hydrolyze ATP are frequently involved in the early steps of DNA replication. Recent studies of Saccharomyces cerevisiae suggest that two members of the AAA+ ATPase family--the origin recognition complex and Cdc6p--have separable roles for ATP binding and ATP hydrolysis during eukaryotic DNA replication. Intriguingly, the proposed regulation of these eukaryotic replication proteins by ATP has functional similarities to the ATP-dependent control of the DnaA and DnaC initiation factors from Escherichia coli. Comparison of the ATP regulation of these factors suggests that ATP binding and hydrolysis acts as a molecular switch that couples key events during initiation of replication. This switch results in a significant change in protein function.

Adenosine Triphosphatases↗
PubMed1997

Interaction of P1 RepA with replication origin of plasmid Rts1: capability of an initiator protein inducing replication from a foreign origin.

Rts1 RepA and P1 RepA are trans-acting proteins essential for the initiation of replication of plasmid Rts1 and prophage P1, respectively. In this study, we found that, in vitro, P1 RepA bound to the Rts1 ori fragment and Rts1 incI fragment as strongly as Rts1 RepA. In addition P1 RepA, in trans, activated the Rts1 replication origin that was cloned in pBR322, thus allowing the ori plasmid to be maintained in a polA E. coli host. Under these conditions, however, the ori plasmid was unstable as compared with that when activated by Rts1 RepA. In addition, we found that Rts1 RepA showed no interaction with the P1 replication origin.

Bacteriophage P1↗
PubMed2002

Noc3p, a bHLH protein, plays an integral role in the initiation of DNA replication in budding yeast.

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.

Blotting, Western↗
PubMed2002

A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidnece from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo.

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.

Adenosine Triphosphate↗