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At least 577 records · Page 32Linked to original sources
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↗
PubMed1995

Replication enhancer requirement for recognition of heterologous replication origin by an initiator protein.

In the pT181 plasmid family, the replication initiation protein (Rep) encoded by each plasmid recognizes only its cognate origin, unless the Rep protein is expressed at abnormally high levels. Heterologous recognition of the origin of the pC221 plasmid by the RepC protein of the pT181 plasmid requires that cmp, the pT181 replication enhancer, be present on the same plasmid as the origin of replication. These findings indicate that cmp has a role in the specificity of Rep-ori recognition and support the model that cmp facilitates the formation/stabilization of the RepC-origin complex.

Bacterial Proteins↗
PubMed2000

Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei.

Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2.

Amino Acid Sequence↗
PubMed1979

Replication and maturation of phage P22 in a mutant of Salmonella typhimurium temperature sensitive in initiation of DNA replication.

TB37 is a dna A-mutant of Salmonella typhimurium in which the initiation of DNA replication at the origin is stopped at 42 degrees C. DNA synthesis in uninfected cells of this strain and in cells infected by phage P22 was followed by the pulse labelling technique. DNA replication ceases completely after about 50 minutes at the high temperature. After lytic infection with P22 (c2) at this time, DNA synthesis starts immediately and increases at a rate well comparable to the permissive control. Obviously the temperature sensitive function of the dnaA-product is dispensable for P22 DNA replication, especially for its initiation. This result is confirmed by the normal yield of phage particles under these conditions, provided that a late step in P22 maturation which naturally is temperature sensitive can proceed at low temperature. If TB37 is infected at 42 degrees C with P22 wild type, an unexpected high rate of phage controlled DNA synthesis is observed. Preliminary results seem to indicate that the process of integration is a prerequisite for part of this synthesis.

DNA Replication↗
PubMed1995

Replication of the broad-host-range plasmid RK2: isolation and characterization of a spontaneous deletion mutant that can replicate in Agrobacterium tumefaciens but not in Escherichia coli.

Two spontaneous deletions of a derivative of the broad-host-range plasmid RK2 were isolated from Agrobacterium tumefaciens. The two deletions have lost 56 and 505 bp, respectively, near the origin of replication (oriV). Of the eight 17-bp repeats present in the RK2 oriV, the smaller deletion has lost the first two while the larger one has lost the first three. The deletions led to a significant increase (3- to 7-fold) in plasmid copy number in A. tumefaciens, indicating their importance in copy number control. While the smaller deletion could replicate in Escherichia coli, the larger one could not. The role of the oriV sequences in the replication of pRK2 in A. tumefaciens and in E. coli is discussed.

Agrobacterium tumefaciens↗
PubMed1990

The cytoplasmic HBVDNA implication: HBV replication or non-replicative status.

Intrahepatocellular HBVDNA was investigated in patients with chronic hepatitis B by in situ hybridization assay, employing biotin-labelled HBVDNA as the specific probe. It was found that HBVDNA was predominantly present in the cytoplasm of hepatocytes. In general the cases with cytoplasmic HBVDNA could be grouped as follows: 1) cytoplasmic HBVDNA coinciding with intrahepatic envelope protein and nucleocapsid component expression, this pattern of the cytoplasmic HBVDNA would be replicative; 2) cytoplasmic HBVDNA accompanied by intrahepatic single HBsAg expression, common to HBVDNA integrated into host genome with only HBsAg being found frequently in liver; 3) with only cytoplasmic HBVDNA, with neither HBsAg nor HBcAg expression, representative for non-replicative status. It is speculated that the reason for the occurrence of the cytoplasmic hybridization signal in cases with non-replicative HBVDNA may be due to DNA-RNA transcript hybrid. The prudent way to evaluate the implication of the cytoplasmic HBVDNA is to consider the combination of HBVDNA location, intrahepatic gene product expression, and HBV markers in the serum.

Cytoplasm↗
PubMed1999

Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication.

Replication of adenovirus (Ad) DNA depends on interactions between three viral and three cellular proteins. Human transcription factors NFI and Oct-1 recruit the Ad DNA polymerase to the origin of DNA replication as a complex with the Ad protein primer pTP. High affinity and specificity DNA binding to recognition sites in this origin by the transcription factors stimulate and stabilize pre-initiation complex formation to compensate for the low binding specificity of the pTP/pol complex. In this review, we discuss the properties of NFI and Oct-1 and the mechanism by which they enhance initiation of DNA replication. We propose a model that describes the dynamics of initiation and elongation as well as the assembly and disassembly of the pre-initiation complex.

Adenoviridae↗
PubMed2004

Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability.

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.

Animals↗
PubMed1972

Replication of bacteriophage M13: specificity of the Escherichia coli dnaB function for replication of double-stranded M13 DNA.

Infection of the temperature-sensitive E. coli mutant HfrH 165/70 (dnaB) with the filamentous single-stranded DNA phage M13 is abortive at the restrictive temperature. Upon infection at 41 degrees , single-stranded phage DNA penetrates the cell and is converted in a rifampicin-sensitive step to the double-stranded replicative form (RF). The parental RF attaches to the cell membrane, but subsequent replication of the RF is blocked. It is concluded that in M13 infection semiconservative RF replication of a double strand to a double strand, in contrast to single-stranded DNA synthesis, depends specifically on the dnaB function.

Centrifugation, Density Gradient↗
PubMed1980

Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13.

The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid plasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected polA- Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare transducing phages carrying single-stranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmids containing a 270-nucleotide fragment from the gene II-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequence coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M13-infected polA- cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA.

Coliphages↗
PubMed1996

Dissection of functional domains of the human DNA replication protein complex replication protein A.

Replication protein A (RPA) is a mammalian single-stranded DNA binding factor essential for DNA replication, repair, and recombination. It is composed of three subunits of 70, 34, and 13 kDa (Rpa1, Rpa2, and Rpa3, respectively). Deletion mapping of the Rpa2 subunit identified the domain required for interaction with Rpa1 and Rpa3 which does not include the N-terminal domain that is phosphorylated during S phase. Deletion mapping of Rpa1 defined three domains. The C-terminal third of the Rpa1 polypeptide binds Rpa2 which itself forms a bridge between Rpa1 and Rpa3. The N-terminal third of Rpa1 bound single-stranded DNA under low stringency conditions only (0.1 M NaCl), while a central domain binds to single-stranded DNA under both low and high stringency conditions (0.5 M NaCl). Binding to p53 requires the N-terminal third of Rpa1 with some contribution from the C-terminal third. The evolutionarily conserved putative zinc finger near the C terminus of Rpa1 was not required for binding to single-stranded DNA, Rpa2, or p53. However, all three subdomains of Rpa1 and the zinc finger were essential for supporting DNA replication in vitro. These experiments are a first step toward defining peptide components responsible for the many functions of the RPA protein complex.

Animals↗
PubMed1997

Deletion analysis of the large subunit p140 in human replication factor C reveals regions required for complex formation and replication activities.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases delta and epsilon. RFC contains multiple activities, including its ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template allowing processive DNA chain elongation. Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36), and RFC activity can be reconstituted from the five cloned gene products. To characterize the role of the large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required for complex formation with the four small subunits. Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of an RFC complex with enhanced activity in replication and PCNA loading. Deletion of additional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupted RFC replication function. DNA primer end recognition and PCNA binding activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished.

DNA↗
PubMed2002

On the specificity of interaction between the Saccharomyces cerevisiae clamp loader replication factor C and primed DNA templates during DNA replication.

Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K(d) approximately 10 nm). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k(off) > 0.2 s(-1) versus 0.025 s(-1), respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly.

Binding Sites↗
PubMed1997

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E. coli cells.

Adenosine Diphosphate↗
PubMed1999

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.

DNA Replication↗
PubMed2002

Site-specific ORC binding, pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins.

Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences. In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa.

Base Composition↗
PubMed1996

The large subunit of replication factor C (Rfc1p/Cdc44p) is required for DNA replication and DNA repair in Saccharomyces cerevisiae.

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

Base Sequence↗
PubMed1986

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.

Animals↗