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PubMed1995

The DNA ligands influence the interactions between the herpes simplex virus 1 origin binding protein and the single strand DNA-binding protein, ICP-8.

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.

Base Sequence↗

PubMed1985

The complete 30-base-pair origin region of bacteriophage phi X174 in a plasmid is both required and sufficient for in vivo rolling-circle DNA replication and packaging.

The origin of replication of the isometric single-stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling-circle DNA replication and packaging of single-stranded (ss) plasmid DNA into phage coats in phi X174 or G4-phage-infected cells. This system was used to study the nucleotide sequence requirements for rolling-circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30-base-pair (bp) origin region of phi X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30-bp origin region and plasmids carrying the 30-bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of phi X174 replicative-form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30-bp origin region and that no contribution is made by sequences which immediately surround the origin region in the phi X174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30-bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the phi X174 and G4 systems respectively). Previous studies [Fluit, A.C., Baas, P.D., van Boom, J.H., Veeneman, G.H. and Jansz, H.S. (1984) Nucleic Acids Res. 12, 6443--6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by phi X174 gene A protein. Moreover, Brown et al. [Brown, D.R., Schmidt-Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402--8412] have shown that omission of the last 2 bp of the origin region does not interfere with phi X174 rolling-circle DNA replication in vitro. Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for phi X174 phage DNA replication and morphogenesis.

Bacteriophage phi X 174↗

PubMed1995

E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration.

The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.

Bacterial Outer Membrane Proteins↗

PubMed1983

Alignment of the genome of monkey B-lymphotropic papovavirus to the genomes of simian virus 40 and BK virus.

We located the origin of DNA replication of African green monkey B-lymphotropic papovavirus DNA by analyzing pulse-labeled form I DNA. With the replication origin used as a reference point, the B-lymphotropic papovavirus genome was aligned with the genomes of simian virus 40 and BK virus from DNA homology between specific fragments hybridized under low-stringency conditions. From the results of these experiments, it was possible to deduce the correlation between the physical and functional maps of the B-lymphotropic papovavirus genome.

BK Virus↗

PubMed2002

Drosophila minichromosome maintenance 6 is required for chorion gene amplification and genomic replication.

Duplication of the eukaryotic genome initiates from multiple origins of DNA replication whose activity is coordinated with the cell cycle. We have been studying the origins of DNA replication that control amplification of eggshell (chorion) genes during Drosophila oogenesis. Mutation of genes required for amplification results in a thin eggshell phenotype, allowing a genetic dissection of origin regulation. Herein, we show that one mutation corresponds to a subunit of the minichromosome maintenance (MCM) complex of proteins, MCM6. The binding of the MCM complex to origins in G1 as part of a prereplicative complex is critical for the cell cycle regulation of origin licensing. We find that MCM6 associates with other MCM subunits during amplification. These results suggest that chorion origins are bound by an amplification complex that contains MCM proteins and therefore resembles the prereplicative complex. Lethal alleles of MCM6 reveal it is essential for mitotic cycles and endocycles, and suggest that its function is mediated by ATP. We discuss the implications of these findings for the role of MCMs in the coordination of DNA replication during the cell cycle.

Amino Acid Sequence↗

PubMed1999

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Adenosine Triphosphate↗

PubMed2004

Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP.

Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5'TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5'TG/TGGATCAG/A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.

Adenosine Diphosphate↗

PubMed1990

Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA.

To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.

Base Sequence↗

PubMed1981

Sequencing long DNA fragments cloned in bacteriophage M13 by using internal primers. The sequence analysis of a yeast DNA fragment containing a replication origin.

In the ;shotgun' procedure for sequencing DNA, DNA fragments are cloned into a phage M13 vector and sequenced by using a flanking primer. In a variation of this procedure a longer DNA sequence is cloned into M13, the two single-stranded recombinants identified and sequenced by using a set of internal primers prepared by exonuclease III digestion of restriction fragments.

Base Sequence↗

PubMed1998

The central lysine in the P-loop motif of the Escherichia coli DnaA protein is essential for initiating DNA replication from the chromosomal origin, oriC, and the F factor origin, oriS, but is dispensable for initiation from the P1 plasmid origin, oriR.

The Escherichia coli DnaA protein is essential for initiation of DNA replication from the chromosomal origin, oriC, and from certain plasmid origins such as oriR of P1, oriS of F, and ori of pSCS101. The DnaA protein binds ATP with high affinity and contains a P-loop motif assumed to be the binding site. Three mutations in the E. coli dnaA gene were constructed by oligonucleotide-directed mutagenesis that changed amino acids in the P-loop. A DnaA protein, K178T, in which the central lysine was changed to the smaller amino acid threonine, was able to initiate DNA replication from P1 oriR, but was unable to initiate replication from E. coli oriC or F oriS in vivo. Mutant and wild-type DnaA proteins were overexpressed, partially purified, and tested for replication activity in vitro. The K178T DnaA protein could initiate replication from oriR, although with a decreased activity compared to the wild-type DnaA protein. No replication activity was detected for this mutant protein from oriC. The different responses of the oriR and oriC replicons to the K178T DnaA protein indicate that the role of DnaA is different in the two systems.

Bacterial Proteins↗

PubMed1997

Binding of a novel host factor to the pT181 plasmid replication enhancer.

Replication enhancers are cis-acting genetic elements that stimulate the activity of origins of DNA replication. The enhancer found in plasmid pT181 of Staphylococcus aureus, called cmp, functions at a distance of 1 kb from the origin of DNA replication to stimulate the interaction between the replication initiation protein and the origin. DNA encoding cmp-binding activity was isolated by screening an expression library of S. aureus DNA in Escherichia coli, and a novel gene, designated cbf1, was identified. The cbf1 locus codes for a polypeptide of 313 amino acid residues (cmp-binding factor 1 [CBF1]; Mr = 35,778). In its COOH-terminal region, the protein sequence contains the helix-turn-helix motif common to many DNA binding proteins that usually bend DNA. The specificity of CBF1 binding for cmp was demonstrated by affinity chromatography using cmp DNA and by competition binding studies. DNase I footprinting analysis of the CBF1-cmp complexes revealed DNase I-hypersensitive sites in phase with the helical periodicity of DNA, implying that CBF1 increases distortion of the intrinsically bent cmp DNA.

Amino Acid Sequence↗

PubMed1996

An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast.

Origins of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle. Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC), which is essential for DNA replication in vivo. Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuclease protection overlapping the ORC footprint. We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo, is essential for the establishment and maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs. These results suggest that pre-RCs play an essential role in initiating DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.

Cell Cycle Proteins↗

PubMed1996

Role for a Xenopus Orc2-related protein in controlling DNA replication.

The six-subunit origin recognition complex (ORC) is essential for the initiation of DNA replication at specific origins in the budding yeast Saccharomyces cerevisiae. An important issue is whether DNA replication in higher eukaryotes, in which the characteristics of replication origins are poorly defined, occurs by an ORC-dependent mechanism. We have identified a Xenopus laevis Orc2-related protein (XORC2) by its ability to rescue a mitotic-catastrophe mutant of the fission yeast Schizosaccharomyces pombe. We show that immunodepletion of XORC2 from Xenopus egg extracts abolishes the replication of chromosomal DNA but not elongation synthesis on a single-stranded DNA template. Indirect immunofluorescence indicates that XORC2 binds to chromatin well before the commencement of DNA synthesis, and even under conditions that prevent the association of replication licensing factor(s) with the DNA. These findings suggest that Orc2 plays an important role at an early step of chromosomal replication in animal cells.

Amino Acid Sequence↗

PubMed1986

Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome.

Early in the staged initiation of enzymatic replication of plasmids containing the unique origin of the E. coli chromosome (oriC), the plasmid is converted to a new topological form which is highly underwound, two to 15 times more than native supercoiled DNA. The underwinding reaction precedes priming of DNA synthesis and follows an initial complex formation, requiring ATP and proteins dnaA, dnaB, and dnaC; underwinding depends on the further addition of gyrase and SSB. DnaB protein as a helicase and gyrase as a topoisomerase drive the underwinding with the energy of ATP hydrolysis. The underwound template, extensively single-stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme.

Bacterial Proteins↗

PubMed1980

Origin-defective mutants of SV40.

We have described a new technique for the isolation and propagation of nonconditionally lethal mutants. We have used this method to generate mutants of SV40 that contain a defective origin of DNA replication. Using these mutants, we have established the following: (1) SV40 DNA replication and early transcription are functionally separate. (2) A functional viral origin of DNA replication is not necessary for the maintenance of transformation. (3) The lack of the origin of SV40 DNA replication does not affect the efficiency of transformation when nonpermissive cells are transfected by DNA using the calcium technique.

Animals↗

PubMed2003

The 'ORC cycle': a novel pathway for regulating eukaryotic DNA replication.

The function of the 'origin recognition complex' (ORC) in eukaryotic cells is to select genomic sites where pre-replication complexes (pre-RCs) can be assembled. Subsequent activation of these pre-RCs results in bi-directional DNA replication that originates at or close to the ORC DNA binding sites. Recent results have revealed that one or more of the six ORC subunits is modified during the G1 to S-phase transition in such a way that ORC activity is inhibited until mitosis is complete and a nuclear membrane is assembled. In yeast, Cdk1/Clb phosphorylates ORC. In frog eggs, pre-RC assembly destabilizes ORC/chromatin sites, and ORC is eventually hyperphosphorylated and released. In mammals, the affinity of Orc1 for chromatin is selectively reduced during S-phase and restored during early G1-phase. Unbound Orc1 is ubiquitinated during S-phase and in some cases degraded. Thus, most, perhaps all, eukaryotes exhibit some manifestation of an 'ORC cycle' that restricts the ability of ORC to initiate pre-RC assembly to the early G1-phase of the cell cycle, making the 'ORC cycle' the premier step in determining when replication begins.

Animals↗

PubMed1992

Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance.

We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.

Autoradiography↗

PubMed1984

A size analysis of the adenovirus replicon.

The linear double-stranded genome of adenovirus DNA replicates semiconservatively from two origins of replication at either of the two molecular ends. Using an in vitro replication system which is able to initiate de novo DNA synthesis we have mapped the origin of DNA replication within the terminal 19 bp of the viral genome. Our conclusions are based on the use of different natural DNA templates, i.e., adenovirus type 2 and mouse adenovirus Fl DNA. In addition, we have employed linearized plasmid DNA templates which contain cloned terminal restriction enzyme fragments as well as chemically synthesized adenovirus termini of different length.

Adenoviridae↗