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At least 397 records · Page 22Linked to original sources
PubMed1992

Topoisomerase I is preferentially associated with normal SV40 replicative intermediates, but is associated with both replicating and nonreplicating SV40 DNAs which are deficient in histones.

Based on the use of equilibrium centrifugation in CsCl to separate covalent complexes between topoisomerase I and DNA from protein-free DNA, it was concluded previously that the topoisomerase is preferentially associated with replicating SV40 DNA (Champoux, J. J. 1988. J. Virol. 62:3675-3683). One explanation for the failure to find the enzyme associated with nonreplicating viral DNA is that most of the completed DNA is rapidly sequestered for encapsidation and inaccessible to topoisomerase I. This explanation has been ruled out in the present work by the finding that topoisomerase I in COS-1 cells is also preferentially associated with the replicative form of an SV40 origin-containing plasmid that lacks the genes coding for the virion structural proteins and therefore cannot be encapsidated. Thus it appears that some structural feature of the replicating DNA or the replication complex specifically recruits the topoisomerase to the DNA. SV40 DNA which is produced in the presence of the protein synthesis inhibitor, puromycin, is deficient in histones and as a result lacks normal chromatin structure. Topoisomerase I was found to be associated with SV40 DNA under these conditions whether or not it was replicating. This observation is interpreted as an indication that under normal conditions, chromatin structure limits access of topoisomerase I to the nonreplicating viral DNA.

Animals↗
PubMed1978

Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication.

Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction.

DNA Replication↗
PubMed2005

A study on the correlation of nucleotide skews and the positioning of the origin of replication: different modes of replication in bacterial species.

Deviations from Chargaff's 2nd parity rule, according to which A approximately T and G approximately C in single stranded DNA, have been associated with replication as well as with transcription in prokaryotes. Based on observations regarding mainly the transcription-replication co-linearity in a large number of prokaryotic species, we formulate the hypothesis that the replication procedure may follow different modes between genomes throughout which the skews clearly follow different patterns. We draw the conclusion that multiple functional sites of origin of replication may exist in the genomes of most archaea and in some exceptional cases of eubacteria, while in the majority of eubacteria, replication occurs through a single fixed origin.

Archaea↗
PubMed2001

The structure and function of the replication initiator protein (Rep) of pSC101: an analysis based on a novel positive-selection system for the replication-deficient mutants.

Plasmid pSC101 encodes a 37.5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to initiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini(-)) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini(-) mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.

Bacterial Proteins↗
PubMed1998

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921.

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.

Amino Acid Sequence↗
PubMed2006

Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity.

The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.

Conjugation, Genetic↗
PubMed1973

Studies on the mechanism of replication of adenovirus DNA. II. The nature of single-stranded DNA in replicative intermediates.

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.

Adenosine Triphosphate↗
PubMed1974

Role of genetic recombination in DNA replication of bacteriophage lambda. II. Effect in DNA replication by gene delta.

We have studied the effect of delta mutations in phage lambda on DNA synthesis as assayed by the accumulation of lambda DNA in infected cells. We find that delta mutants appear to generate somewhat less DNA than lambda(+) in a rec(+) host, suggesting the wild-type delta gene may act in DNA replication. An additional clue to delta function arises if replication is measured in the gamma-negative situation where concatemer formation is abortive. In this situation, the wild-type delta gene has an "inhibitory" effect on replication. A similar inhibitory effect on replication due to delta is observed after infection of P(2) lysogens. We conclude from these studies that the delta gene may act with alpha, beta, and gamma genes, possibly in a process affecting DNA replication.

Coliphages↗
PubMed1977

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).

Cell Line↗
PubMed1991

Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.

Adenoviruses, Human↗
PubMed1997

Specific initiation of replication at the right-end telomere of the closed species of minute virus of mice replicative-form DNA.

We have developed an in vitro system that supports the replication of natural DNA templates of the autonomous parvovirus minute virus of mice (MVM). MVM virion DNA, a single-stranded molecule bracketed by short, terminal, self-complementary sequences, is converted into double-stranded replicative-form (RF) DNA when incubated in mouse A9 fibroblast extract. The 3' end of the newly synthesized complementary strand is ligated to the right-end hairpin of the virion strand, resulting in the formation of a covalently closed RF (cRF) molecule as the major conversion product. cRF DNA is not further replicated in A9 cell extract alone. On addition of purified MVM nonstructural protein NS1 expressed from recombinant baculoviruses or vaccinia viruses, cRF DNA is processed into a right-end (5' end of the virion strand) extended form (5'eRF). This is indicative of NS1-dependent nicking of the right-end hairpin at a distinct position, followed by unfolding of the hairpin and copying of the terminal sequence. In contrast, no resolution of the left-end hairpin can be detected in the presence of NS1. In the course of the right-end nicking reaction, NS1 gets covalently attached to the right-end telomere of the DNA product, as shown by immunoprecipitation with NS1-specific antibodies. The 5'eRF product is the target for additional rounds of NS1-induced nicking and displacement synthesis at the right end, arguing against the requirement of the hairpin structure for recognition of the DNA substrate by NS1. Further processing of the 5'eRF template in vitro leads to the formation of dimeric RF (dRF) DNA in a left-to-left-end configuration, presumably as a result of copying of the whole molecule by displacement synthesis initiated at the right-end telomere. Formation of dRF DNA is highly stimulated by NS1. The experimental results presented in this report support various assumptions of current models of parvovirus DNA replication and provide new insights into the replication functions of the NS1 protein.

Animals↗
PubMed1994

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.

Animals↗
PubMed2006

Replication-dependent DNA damage response triggered by roscovitine induces an uncoupling of DNA replication proteins.

The cyclin-dependent kinase (CDK) inhibitor roscovitine is under evaluation in clinical trials for its antiproliferative properties. Roscovitine arrests cell cycle progression in G(1) and in G(2) phase by inhibiting CDK2 and CDK1, and possibly CDK7 and CDK9. However, the effects of CDK2 inhibition in S-phase cells have been not fully investigated. Here, we show that a short-term treatment with roscovitine is sufficient to inhibit DNA synthesis, and to activate a DNA damage checkpoint response, as indicated by phosphorylation of p53-Ser15, replication protein A, and histone H2AX. Analysis of DNA replication proteins loaded onto DNA during S phase showed that the amount of proliferating cell nuclear antigen (PCNA), a cofactor of DNA replication enzymes, was significantly reduced by roscovitine. In contrast, chromatin-bound levels of DNA polymerase delta, DNA ligase I and CDK2, were stabilized. Checkpoint inhibition with caffeine could rescue PCNA disassembly only partially, pointing to additional effects due to CDK2 inhibition and the presence of replication stress. These results suggest that in S-phase cells, roscovitine induces checkpoint-dependent and -independent effects, leading to stabilization of replication forks and an uncoupling between PCNA and PCNA-interacting proteins.

Cell Proliferation↗
PubMed2000

Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts.

We identified a Drosophila gene, double parked (dup), that is essential for DNA replication and belongs to a new family of replication proteins conserved from Schizosaccharomyces pombe to humans. Strong mutations in dup cause embryonic lethality, preceded by a failure to undergo S phase during the postblastoderm divisions. dup is required also for DNA replication in the adult ovary, establishing that dup is needed for DNA replication at multiple stages of development. Strikingly, DUP protein colocalizes with the origin recognition complex to specific sites in the ovarian follicle cells. This suggests that DUP plays a direct role in DNA replication. The dup transcript is cell cycle regulated and is under the control of E2F and Cyclin E. Interestingly, dup mutant embryos fail both to downregulate S phase genes and to engage a checkpoint preventing mitosis until completion of S phase. This could be either because these events depend on progression of S phase beyond the point blocked in the dup mutants or because DUP is needed directly for these feedback mechanisms.

Alleles↗
PubMed1987

Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system.

We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell-free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin-11-dUTP, and isolated nuclei were then probed with fluorescein-conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2-6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that initiations are coordinated within each nucleus such that the nucleus is the fundamental unit of replication in the cell-free system.

Animals↗
PubMed1987

Start sites for bidirectional in vitro DNA replication inside the replication origin, oriC, of Escherichia coli.

In vitro replication of mini-chromosomes in the absence of DNA ligase activity resulted in replication products with single-strand breaks at specific sites. The occurrence of these nicks was coupled to an active replication process, therefore we expect them to represent start sites for DNA replication. Two positions within oriC for each of the two leading strands of bidirectional replication were found. Within each position are one or two start sites. Counterclockwise synthesis started at positions 194/199 and 265/272, clockwise synthesis at positions 209/219 and 254. The start positions are located close to DnaA protein binding sites. A model for initiation accommodating this observation is discussed.

Chromosomes, Bacterial↗
PubMed1994

Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.

The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.

Animals↗
PubMed1995

Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.

The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol. wt 14,500. RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coli system. We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E. coli only when two RTP dimers are bound co-operatively to overlapping core and auxiliary sequences comprising the terminus. A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity. Binding of two RTP dimers to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination. RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length. Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.

Bacillus subtilis↗