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At least 1,225 records · Page 68Linked to original sources
PubMed1999

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus.

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.

Caulobacter crescentus↗
PubMed1997

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315.

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.

Amino Acid Sequence↗
PubMed1983

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.

Antigens, Viral↗
PubMed1996

Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions.

The human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int-) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA-binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S. I) of both viral origin regions, whereas SVT preferentially bound B.S. I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCV in vitro, and to the virus' ability to establish persistent infections in vivo.

Animals↗
PubMed1988

Multiple insertions and tandem repeats of origin-minus simian virus 40 DNA in transformed rat and mouse cells.

Stable simian virus 40 (SV40) transformation requires integration and expression of the early region of the SV40 genome. We have examined the amount and state of integrated viral DNA of SV40-transformed NIH 3T3 mouse and F2408 rat fibroblast lines generated by transfection with either wild-type or origin-defective SV40 DNA. A functional SV40 replication origin was not required for multiple inserts and partial-repeat structures to form in NIH 3T3 mouse transformants. In contrast, partial repeats in F2408 rat transformants were rare when the SV40 replication origin was intact and not detected at all when it was defective.

Animals↗
PubMed1992

The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells.

An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle traverse in the mimosine [corrected]-resistant CHO cell line CHOC 400. When administered to cultures for 14 h after reversal of a G0 block, the drug appears to arrest the population at the G1/S boundary, and upon its removal cells enter the S phase in a synchronous wave. However, when methotrexate is administered to an actively dividing asynchronous culture, cells are arrested not only at the G1/S interface but also in early and middle S phase. Most interestingly, two-dimensional gel analysis of replication intermediates in the initiation locus of the amplified dihydrofolate reductase domain suggests that mimosine may actually inhibit initiation. Thus, this drug represents a new class of inhibitors that may open a window on regulatory events occurring at individual origins of replication.

Animals↗
PubMed1997

RNase mitochondrial RNA processing correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication.

The precursor primer RNA for mammalian mitochondrial DNA leading-strand replication remains as a persistent R loop formed during transcription through the mitochondrial DNA control region. We have examined model R loops, which exist in a novel and physiologically accurate preprimer conformation, as potential substrates for mammalian RNase mitochondrial RNA processing (MRP). Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial DNA origin. The multiple cleavage sites on the R-loop substrate match the priming sites observed in vivo, suggesting that RNase MRP alone is capable of generating virtually all of the leading-strand replication primers.

Animals↗
PubMed1976

Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons.

Although it carries two competent replication systems, a composite plasmid formed in vitro by linkage of the complete ColE1 and pSC101 plasmid replicons at their unique EcoRI endonuclease cleavage sites normally uses only the replication origin and functions of the ColE1 component. Restriction of ColE1 replication functions by DNA polymerase I deprivation results, however, in exclusive use of the pSC101 replication origin. When using the ColE1 replication system the composite plasmid is nevertheless incompatible with both the parent replicons. This suggests that a trans-dominant gene product is involved in plasmid incompatibility and supports negative control rather than positive control models for regulation of the initiation of DNA replication.

DNA Replication↗
PubMed2002

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells.

The mechanism of disease-associated trinucleotide repeat instability involves cis-acting factors (cis-elements) in the vicinity of the repeat, but the nature of these elements is unknown. One cis-element may be the location of the replication origin relative to the repeat. We have used an SV40 DNA replication system to investigate the effect of the location of replication initiation on (CTG)(n)*(CAG)(n) stability in primate cells. Depending on the distance between the SV40 replication origin and the repeat tract, templates with 79 repeats yield predominantly expansions or predominantly deletions or remain intact. All templates with 17 repeats are stable. Thus, cis-elements that affect the sites of Okazaki fragment initiation relative to the repeat are crucial determinants of instability. This model system recapitulates the bias for expansions observed in many of the diseases associated with trinucleotide repeats. Our results might explain the variable amounts of CTG/CAG instability that are observed in different chromosomal contexts.

Animals↗
PubMed1987

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid.

We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA- and ParB- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two proteins whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.

Amino Acid Sequence↗
PubMed2002

Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45.

Current models suggest that the replication initiation factor Mcm10 is required for association of Mcm2-7 with origins of replication to generate the prereplicative complex (pre-RC). Here we report that Xenopus Mcm10 (XMcm10) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcm10 at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcm10 performs its function after pre-RC assembly and before origin unwinding. As one of the earliest known pre-RC activation steps, chromatin binding of XMcm10 is an attractive target for regulation by cell cycle checkpoints.

Animals↗
PubMed1998

Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders.

Expression of the Cdc6 protein (Cdc6p) is essential for formation of prereplicative complexes at budding yeast replication origins. Analysis of mutations in the conserved nucleoside triphosphate (NTP)-binding site of Cdc6p described here suggests that NTPs are required both for the productive interaction of Cdc6p with replication origins during G1 and the quantitative loading of the Mcm2-7 family of proteins onto chromatin. We show that Cdc6p exhibits significant sequence similarity to subunits of eukaryotic and prokaryotic clamp-loaders, which load ring-shaped DNA polymerase processivity factors onto DNA in an analogous reaction. Similarities in both sequence and mechanism suggest that Cdc6p and the clamp-loaders are members of a superfamily of nucleotide-dependent loading factors.

Alleles↗
PubMed1982

A new RNA polymerase and in vitro transcription of the origin of replication from rat mitochondrial DNA.

A new RNA polymerase was found in a rat mitochondrial extract. This enzyme showed strong template preference in vitro for the supercoiled recombinant plasmid consisting of pBR322 and the D-loop region of rat mtDNA carrying the origin of heavy-strand replication. The main products synthesized by the D-loop region were two RNAs of different sizes. Both of these products were light-strand products transcribed from the region upstream from the origin of replication. This specific transcription is discussed in relation to initiation of primer RNA synthesis for heavy-strand replication of rat mtDNA.

Animals↗
PubMed1982

Surrogate origins of replication in the mitochondrial genomes of ori-zero petite mutants of yeast.

We have investigated the mitochondrial genome of eight ori-zero spontaneous petite mutants of Saccharomyces cerevisiae. The tandem repeat units of these genomes do not contain any of the seven canonical ori sequences of the wild-type genome. Instead, they contain one, or more, ori-S sequences. These 44-nucleotide long surrogate origins of replication are a subset of GC clusters characterized by a potential secondary fold with two sequences ATAG and GGAG , inserted in AT spacers, two AT base pairs just following them, a GC stem (broken in the middle, and, in most cases also near the base, by non-paired nucleotides), and a terminal loop. This structure is reminiscent of that of GC clusters A and B from canonical ori sequences and supports the view (Bernardi, 1982a ) that the GC clusters of the mitochondrial genome arose, by an expansion process, from the canonical ori sequences. Like the latter, ori-S sequences are present in both orientations, are located in intergenic regions, and can be used as excision sequences when tandemly oriented. Again as in the case of canonical ori sequences, the density of ori-S sequences on the repeat units of petite genomes are correlated with the replication efficiency of the latter, as assessed by the outcome of crosses with wild-type or petite tester strains.

Base Sequence↗
PubMed1993

Optimalization of southwestern technique for detection of binding of Epstein-Barr virus nuclear antigen 1 to origin of replication.

An optimized protocol of Southwestern analysis for detection of Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) binding to the origin of viral replication (oriP) was described. The unique feature of this optimized protocol includes the restoration of the denatured proteins to native conformation after SDS-polyacrylamide gel electrophoresis and blocking of nonspecific and low-affinity binding sites prior to DNA binding. The parameters and conditions that may affect the specific interaction of EBNA-1 to oriP DNA were then determined. The specific binding was affected by divalent cations (MgCl2) and ionic strength; the optimal concentrations of MgCl2 and NaCl were observed at 10 and 300 mM, respectively. The various buffer systems and pH values tested had no apparent effect on EBNA-1 binding. Under the optimal conditions, a single protein of 68 Kd was detected and the bindings of other nonspecific, low-affinity DNA-binding proteins were abolished. The authenticity and specificity of 68 Kd protein as EBNA-1 were determined by reaction with antibody specific for EBNA-1 and competition assay with specific DNA sequence. After optimization this technique can be a powerful and yet simple means of studying protein-DNA interactions and their roles in gene expression.

Antigens, Viral↗
PubMed1987

Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1.

The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1. Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli. The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S. lividans 66 was determined. All except one of the inserts in the 1.52-kb essential region inactivated replication. A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment. The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.

Base Sequence↗