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PubMed1993

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.

Amino Acid Sequence↗

PubMed1999

Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p stability.

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome↗

PubMed1990

The single-stranded DNA-binding protein of Escherichia coli.

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.

Amino Acid Sequence↗

PubMed1984

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences.

An in vitro system which replicates plasmid DNA containing the replication origin of adenovirus DNA has been established. Replication of plasmid pLA1 DNA, which contains the left-hand terminus (0-9.4 map units) of adenovirus serotype 5 DNA but which lacks the 55,000-dalton terminal protein, is initiated by a protein-primed mechanism in a manner similar to that found with adenovirus DNA. Initiation of DNA replication using plasmid pLA1 as a template requires (i) that the cloned adenovirus sequence be present at the terminus of a linearized (form III) DNA molecule ( Tamanoi , F., and Stillman , B. W. (1982) Proc. Natl. Acad. Sci. U. S. A., 79, 2221-2225; van Bergen, B. G. M., van der Ley , P. A., van Driel , W., van Mansfield , A. D. M., and van der Vliet , P. A. (1983) Nucleic Acid Res. 11, 1975-1979), and (ii) the presence of the 80,000-dalton precursor to the 55,000-dalton terminal protein and the adenovirus coded DNA-dependent DNA polymerase. In the presence of the four deoxy-nucleoside triphosphates, the preterminal protein, the adenovirus coded DNA binding protein, and an extract prepared from uninfected HeLa nuclei, the adenovirus DNA polymerase can elongate the preterminal-protein dCMP initiation complex formed on pLA1 DNA to full length (6.6 kilobase) DNA molecules. These results suggest that the 55,000-dalton terminal protein covalently linked to the 5' termini of adenovirus DNA is not essential for the replication of this DNA.

Adenoviridae↗

PubMed2004

Oscillating global regulators control the genetic circuit driving a bacterial cell cycle.

A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

Bacterial Proteins↗

PubMed1994

The incN plasmid replicon: two pathways of DNA polymerase I-independent replication.

The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected. The requirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can be recovered as a plasmid only if RepA is provided in trans. These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.

Bacterial Proteins↗

PubMedDate not supplied

[DNA fragment DARC146 from a complex form of DNA polymerase alpha contains several nuclear protein binding segments].

Earlier, a number of DNA fragments were identified in the complex form of DNA polymerase alpha. One of them, DARC146, can support autonomous replication in mammalian cells. We have subcloned 146 bp from DARC146 (here called DARC146). This fragment has an ability to replicate autonomously in mammalian cells. This ability permits one to speak about DARC146 as a putative replication origin. From this conclusion, we suggest that all signals for initiation of DNA synthesis are located on the nucleotide sequence under study. Here, we have shown that the nuclear extract contains four polypeptides binding specifically to synthetic oligonucleotides covering the AT-rich region of the DARC146 sequence. The first protein is Oct-1, a nuclear transcription-replication factor. The second protein (named p65) binds to the TCTCTTA site of the DARC146 nucleotide sequence. There are two sites for Oct-1 protein and two sites for p65 in the DARC146 fragment. Octamer motifs and sites for p65 are located tandemly side by side. Moreover, we identified 28kDa polypeptide from nuclear matrix which bound to DARC146. Based upon the data presented, we suggest a hypothetical model of the pre-initiation state of the DARC146 sequence.

Animals↗

PubMed2002

RacA, a bacterial protein that anchors chromosomes to the cell poles.

Eukaryotic chromosomes are anchored to a spindle apparatus during mitosis, but no such structure is known during chromosome segregation in bacteria. When sister chromosomes are segregated during sporulation in Bacillus subtilis, the replication origin regions migrate to opposite poles of the cell. If and how origin regions are fastened at the poles has not been determined. Here we describe a developmental protein, RacA, that acts as a bridge between the origin region and the cell poles. We propose that RacA assembles into an adhesive patch at a centromere-like element near the origin, causing chromosomes to stick at the poles.

Bacillus subtilis↗

PubMed1998

DnaB helicase is unable to dissociate RNA-DNA hybrids. Its implication in the polar pausing of replication forks at ColE1 origins.

A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.

Autoradiography↗

PubMed2002

Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex.

In eukaryotic cells, an ordered sequence of events leads to the initiation of DNA replication. During the G(1) phase of the cell cycle, a prereplication complex (pre-RC) consisting of ORC, Cdc6, Cdt1, and MCM2-7 is established at replication origins on the chromatin. At the G(1)/S transition, MCM10 and the protein kinases Cdc7-Dbf4 and Cdk2-cyclin E cooperate to recruit Cdc45 to the pre-RC, followed by origin unwinding, RPA binding, and recruitment of DNA polymerases. Using the soluble DNA replication system derived from Xenopus eggs, we demonstrate that immunodepletion of protein phosphatase 2A (PP2A) from egg extracts and inhibition of PP2A activity by okadaic acid abolish loading of Cdc45 to the pre-RC. Consistent with a defect in Cdc45 loading, origin unwinding and the loading of RPA and DNA polymerase alpha are also inhibited. Inhibition of PP2A has no effect on MCM10 loading and on Cdc7-Dbf4 or Cdk2 activity. The substrate of PP2A is neither a component of the pre-RC nor Cdc45. Instead, our data suggest that PP2A functions by dephosphorylating and activating a soluble factor that is required to recruit Cdc45 to the pre-RC. Furthermore, PP2A appears to counteract an unknown inhibitory kinase that phosphorylates and inactivates the same factor. Thus, the initiation of eukaryotic DNA replication is regulated at the level of Cdc45 loading by a combination of stimulatory and inhibitory phosphorylation events.

Carrier Proteins↗

PubMed1991

The mutH gene regulates the replication and methylation of the pMB1 origin.

DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColE1 origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMB1 origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMB1 replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.

Bacterial Proteins↗

PubMed1982

Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.

DNA Polymerase I↗

PubMed1978

Construction of a viable SV40 variant containing two functional origins of DNA replication.

Viable variants of simian virus 40 (SV40) have been constructed which contain two functional origins of DNA replication (Or). The variants were made by introducing, at 0.175 on the SV40 map, a segment of DNA containing the viral Or. Two types of experiments demonstrate that the second Or is functional. First, the distribution of radioactivity in pulse-labeled SV40 (I) DNA is dramatically altered in the variants when compared with the parental virus. Second, electron microscopic examination of viral replicative intermediates indicates that while there is one initiation site for DNA synthesis in the parental genome, there are two sites in the variant. It was possible to introduce a deletion which inactivated the original Or at 0.67 map units in this variant. The resulting mutant could be propagated, and its DNA replication originated at the site of the newly inserted Or.

DNA Replication↗

PubMed1998

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution.

BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion. CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.

Crystallography, X-Ray↗

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↗

PubMed1997

Mammalian artificial chromosomes--vectors for somatic gene therapy.

Mammalian artificial chromosomes might prove to be useful vectors for somatic gene therapy. The functional elements of such an artificial chromosome are telomeres, a centromere and a replication origin. Recent progress in the characterization of these functional elements of the eukaryotic chromosome will be described. Attempts to construct artificial chromosomes for mammalian cells and their use for somatic gene therapy are discussed.

Animals↗

PubMed2004

Modular structure of the human lamin B2 replicator.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Base Sequence↗

PubMed1986

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2 phi X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post-transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA.

Animals↗