Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Replisomes”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed1992

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.

Amino Acid Sequence↗
PubMed2026

Maintenance of nucleosome organization through replication and transcription counteracts aberrant coalescence of active chromatin.

Nucleosomes with their associated modifications organize and regulate the genome. It is unclear how this is integrated with the requirement of replication and transcription to access the DNA template without jeopardizing chromatin function. Here, we reveal a unified requirement for the histone chaperone FACT in mediating nucleosome disruption and reassembly during mammalian replication and transcription. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome wide, and chromatin structure in their wake collapses, with reduced nucleosome occupancy, irregular spacing, and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. Similarly, aberrant compartments form in cells failing to maintain chromatin fiber structure through replication. Nucleosome organization therefore dynamically regulates genome architecture, guarding against spurious chromatin aggregation.

Nucleosomes↗
PubMed2006

Replication fork blockage by transcription factor-DNA complexes in Escherichia coli.

All organisms require mechanisms that resuscitate replication forks when they break down, reflecting the complex intracellular environments within which DNA replication occurs. Here we show that as few as three lac repressor-operator complexes block Escherichia coli replication forks in vitro regardless of the topological state of the DNA. Blockage with tandem repressor-operator complexes was also observed in vivo, demonstrating that replisomes have a limited ability to translocate through high affinity protein-DNA complexes. However, cells could tolerate tandem repressor-bound operators within the chromosome that were sufficient to block all forks in vitro. This discrepancy between in vitro and in vivo observations was at least partly explained by the ability of RecA, RecBCD and RecG to abrogate the effects of repressor-operator complexes on cell viability. However, neither RuvABC nor RecF were needed for normal cell growth in the face of such complexes. Holliday junction resolution by RuvABC and facilitated loading of RecA by RecF were not therefore critical for tolerance of protein-DNA blocks. We conclude that there is a trade-off between efficient genome duplication and other aspects of DNA metabolism such as transcriptional control, and that recombination enzymes, either directly or indirectly, provide the means to tolerate such conflicts.

Bacterial Proteins↗
PubMed1985

DNA damage and cell killing. Cause and effect?

The evidence supporting a cause and effect relationship between DNA damage and cell killing is examined in the light of what is currently known about the organization and replication of genomic DNA in eukaryotic cells and the radio-energetics of DNA breakage. A large disparity is identified between characteristic doses for cell killing and for the production of DNA lesions (i.e., single- or double-strand breaks). In contrast, the sensitive phase of the inhibition of DNA synthesis has a dependence on dose quantitatively similar to that of cell killing. A model is developed in which single- and double-strand breaks are associated with the inhibition of replicon initiation, whereas only double-strand breaks are primarily responsible for strand elongation. Furthermore, the model points to the replisome and the region of replicated DNA just downstream from the fork as the locus of radiation action.

Animals↗
PubMed1984

Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication.

We have determined the DNA sequence and transcription patterns in a 3-kb segment (between 15 and 18 kb on the standard phage T4 map) spanning an origin of DNA replication. A new gene, 69, spans this origin. Gene 69 codes for two overlapping proteins that share a common C-terminal segment. Defective DNA replication in an appropriate amber mutant shows that at least the larger of the two proteins is required for efficient T4 DNA replication. The two proteins coded by gene 69 are expressed from different transcripts that are under different regulation. The smaller protein, gp69*, can be expressed immediately from an Escherichia coli-like promoter, whereas expression of the larger protein, gp69, must be delayed since its middle promoter requires T4 coded proteins, most likely gp mot, for activation. We discuss the possible significance of two overlapping proteins in the assembly of replisomes. Gene 69 is bracketed by the non-essential early gene dam (DNA adenine methylase) and the late gene soc (small outer capsid protein). Transcripts through this region are interdigitated in a complex pattern, which reveals all elements that are thought to be important in regulation of pre-replicative and post-replicative T4 genes.

Amino Acid Sequence↗
PubMed1986

Initiation of Escherichia coli minichromosome replication at oriC and at protein n' recognition sites. Two modes for initiating DNA synthesis in vitro.

The start sites for leading and lagging DNA strands were determined in vitro with minichromosomes as templates. Fragments from replication intermediates were analyzed by hybridization to single-stranded probes. Leading strand synthesis in the counterclockwise direction was found to originate in or close to (position 248 to -44) the minimal origin. Complementary lagging strand synthesis started several positions to the left outside of oriC. The results suggest in addition a concerted synthesis of leading and lagging strands following the dnaA directed assembly of initiation proteins at double-stranded oricC DNA (pre-replisome). In addition, DNA synthesis could initiate at protein n' recognition sequences located within and clockwise to the asnA gene. Initiation at n' sites was dependent on protein i activity, whereas leading and lagging strand initiation in the oriC region was not affected by protein i. Our results argue against an involvement of the phi X174-type primosome in the initiation of discontinuous DNA synthesis at oriC. An alternative function is suggested.

Bacterial Proteins↗
PubMed1991

Ultrastructural cryoimmunocytochemistry is a convenient tool for the study of DNA replication in cultured cells.

In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minutes. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.

Animals↗
PubMed2006

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity.

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

Crystallization↗
PubMed1994

The Bacillus subtilis replication terminator system functions in Escherichia coli.

The Bacillus subtilis DNA terminators, IRI + IRII, were inserted into the Escherichia coli plasmid pACYC184 such that the IRI terminator would be in its active orientation with respect to the approaching unidirectionally moving replication fork. When this new plasmid was transferred into E. coli, harbouring an expression plasmid producing the B. subtilis terminator protein RTP, fork arrest was observed to occur at the position of the inserted terminator region. Thus, the B. subtilis replication terminator system can function in E. coli. It was shown that the B. subtilis system operated with approximately 30% of the efficiency of the E. coli system utilizing the R6K TerR2 DNA terminator and the E. coli Tus terminator protein. Assuming that RTP and Tus have quite different folded structures these results suggest that fork arrest in B. subtilis is not dependent upon a highly specific recognition and interaction between RTP positioned on the DNA terminator and a component(s) of the approaching replisome.

Bacillus subtilis↗
PubMed1998

Replication of R6K gamma origin in vitro: discrete start sites for DNA synthesis dependent on pi and its copy-up variants.

The regulation of the plasmid R6K gamma origin (gamma ori) is accomplished through the ability of the pi protein to act as an initiator and inhibitor of replication. Hyperactive variants of this protein, called copy-up pi, allow four to tenfold increases of gamma ori plasmid DNA in vivo. The higher activity of copy-up pi variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replication that can be used by wt pi (pi35. 0) only under certain conditions. We have compared the replication activities of wt pi35.0 and copy-up pi mutants in vitro, and analyzed the replication products. It is shown that copy-up variants are several-fold more active than wt pi35.0 in replication. This appears to be due to enhanced specific replication activity of copy-up mutants rather than elevated fractions of protein proficient in DNA binding. Furthermore, biochemical complementation revealed that pi200 (copy-up) is dominant over wt pi35.0. The elevated activity of copy-up pi is not caused by an increased rate of replisome assembly as inferred from in vitro replication assays in which the lag periods observed were similar to that of wt pi35.0. Moreover, only one round of semiconservative, unidirectional replication occurred in all the samples analyzed indicating that copy-up pi proteins do not initiate multiple rounds of DNA synthesis. Rather, a larger fraction of DNA template replicates in the presence of copy-up pi as determined by electron microscopy. Two clusters of discrete DNA synthesis start sites are mapped by primer extension near the stability (stb) locus of the gamma ori. We show that the start sites are the same in the presence of wt pi35.0 or copy-up proteins. This comparative analysis suggests that wt pi35.0 and copy-up variants utilize fundamentally similar mechanism(s) of replication priming.

Amino Acid Substitution↗
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↗
PubMed2001

Role of the double-strand origin cruciform in pT181 replication.

pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts the plasmid's double-strand origin (DSO) and extrudes a cruciform involving IR II, a palindrome flanking the initiation nick site. We have hypothesized that the cruciform is required for initiation, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBO J. 16, 4456-4566). In this study, we have tested the requirement for cruciform extrusion by disrupting the symmetry of the IR II palindrome or by increasing its length. The modified DSOs were tested for replication with RepC in trans. Rather surprisingly, disruption of the IR II symmetry had no detectable effect on replication or on competitivity of the modified DSO, though plasmids with IR II disrupted were less efficiently relaxed than the wild type by RepC. However, in conjunction with IR II disruption, modification of the tight RepC binding site IR III blocked replication. These results define two key elements of the pT181 initiation mechanism--the IR II conformation and the RepC binding site (IR III)--and they indicate that pT181 replication initiation is sufficiently robust to be able to compensate for significant modifications in the configuration of the DSO.

Bacterial Proteins↗
PubMed1997

A novel mechanism of virus-virus interactions: bacteriophage P2 Tin protein inhibits phage T4 DNA synthesis by poisoning the T4 single-stranded DNA binding protein, gp32.

P2 prophages have been known to inhibit DNA replication and growth of T-even phages. We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2 tin gene. Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely prevent de novo DNA replication and growth of wild-type T2 or T4 phage. We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein. In all of these mutants, which we call asp, Asp codon 163 of gene 32 is changed to a Gly or Asn codon. The mutant alleles are recessive; i.e., when wild-type and asp mutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein. To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene 32 protein on single-stranded DNA. Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome. The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication.

Bacteriophage P2↗
PubMed2000

Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutant proteins: correlation with in vivo function.

The DNA polymerase (pol) catalytic subunit of herpes simplex virus type 1, encoded by UL30, and its accessory factor, UL42 protein, are both essential for the replication of the virus. Because the stable interaction between UL42 and pol renders the pol fully processive for replicative DNA synthesis, disruption of this interaction represents a potential goal in the development of novel antiviral compounds. To better compare the effects of mutations in UL42 protein on its known in vitro functions, mutations were expressed as glutathione-S-transferase (GST)-fusions and the fusion proteins used in affinity chromatography. In this report, we demonstrate the relationship between the abilities of mutant UL42 fusion proteins to bind pol and to stimulate pol activity in vitro, and the abilities of nonfusion mutant proteins to function in viral replication. The pol stimulation assay using GST fusion proteins was found to be a more accurate and sensitive measure of the ability of the UL42 protein to function in vitro than the pol binding assay using the fusion proteins linked to a solid matrix. We also found an excellent correlation between the ability of purified GST fusion proteins to stimulate pol activity in vitro and the ability of full-length nonfusion UL42 mutant genes to support DNA replication in infected cells. Our results demonstrate that two noncontiguous stretches of amino acids, from 137 to 142 and from 274 to 282, are essential for UL42 function in vivo and in vitro. Although mutant d241-261 exhibited close to wild-type abilities to stimulate pol activity in vitro, it was not capable of complementing the replication of a UL42 null mutant virus. The region of UL42 protein within or close to 241-261 may serve to hinge the essential regions within the N- and C-terminal portions of the protein which are thought to interdigitate. It is hypothesized that reduction in the length of the hinge region could alter the ability of UL42, and/or its complex with pol, to function with one or more of the other proteins present in the DNA replisome within infected cells.

Animals↗
PubMed1985

Abnormal fibroblast aging and DNA replication in the Werner syndrome.

Cell and DNA replicative potentials were studied in 10 strains of skin fibroblasts from unrelated patients with the Werner syndrome (WS) and in a progeric CRL 1277 strain. The lifespans of all WS strains and of CRL 1277 cells are greatly abbreviated in vitro, due to large fractions of non-cycling cells and the basic process of progressive clonal attenuation during Phase II. In some WS strains, the population-doubling rate per day and the cloning efficiency fluctuated concurrently in random fashion during the cellular aging process, indicating alternating successions of adaptively well and poorly growing clones, probably resulting from various chromosome translocations. No detectable defect in excision repair was found in WS or CRL 1277 cells. However, the rate of increase in the molecular weight of pulse-chased DNA involving overall rates of chain elongation and replicon fusion was retarded in WS and CRL 1277 cells. Also, pulse-labelled DNA in WS fibroblasts was less enriched in the nuclear matrix and was more slowly chased out than in normal cells. These results led us to postulate a misfiring or delayed initiation due to the sticky attachment of replicating DNA to the nuclear matrix in the replisomes of WS fibroblasts. A suggested model for abnormal DNA replication is presented and discussed to explain the loss of DNA and the chromosome abnormalities in WS cells. The abnormal DNA-synthetic profiles so derived appeared to be normalized in SV40-transformed PSV811 (WS) cells as were in gamma ray-transformed wild-type WI38CT-1 cells.

Adolescent↗
PubMed1984

Enzyme studies of replication of the Escherichia coli chromosome.

Progress of the replication forks of the Escherichia coli chromosome depends on a multisubunit DNA polymerase (for chain elongation) and a primosome (for chain initiations), together comprising about 30 polypeptides with a mass in excess of 10(6) daltons. Integration of their actions with those of helicases and DNA binding proteins suggest a more complex and integrated replisome assembly with novel possibilities for concurrent replication of both parental strands. Initiation of a new cycle of chromosome replication at its unique 245-bp (oriC) is being studied in a soluble enzyme system with plasmids, autonomous replication of which depends on the oriC sequence. Required proteins include RNA polymerase, DNA gyrase, dnaA protein (with 4 strong binding sites in oriC), HU protein, and additional proteins (e.g., topoisomerase I and ribonuclease H) that confer oriC specificity by suppressing initiation of replication elsewhere on the duplex DNA. Clarification of the biochemical mechanisms of replication is fundamental for understanding cell growth and development. Knowledge of the biochemistry of initiating a cycle of chromosome replication opens the way toward exploring the regulation of the cell cycle. I remain faithful to the conviction that anything a cell can do, a biochemist should be able to do. He should do it even better, being freed from the constraints of substrate and enzyme concentrations, pH, ionic strength, and temperature, and by having the license to introduce novel reagents to drive or restrain a reaction. Put another way, one can be creative more easily with a reconstituted system. One can grapple directly with the molecules instead of trying by remote means to manipulate their structures or levels in the intact cell. Enzyme purification carries many dangers beyond the well-known exposure of the fragile enzyme to the hostilities of an unfamiliar environment, high dilution, glass containers and a denaturable investigator. But the rewards of enzyme purification have justified the effort. The polymerases, nucleases, ligases purified out of curiosity about the mechanisms of replication, repair and recombination have supplied the cast of actors responsible for the current drama of genetic engineering. Beyond the uses of these enzymes as reagents, understanding the mechanisms of DNA metabolism will have practical value in manipulating the replication of plasmids and viruses and the expression of their genes and, beyond that, in obtaining a more secure grasp of chromosome structure and function.

Bacterial Proteins↗
PubMed1995

DNA polymerase delta is required for the replication feedback control of cell cycle progression in Schizosaccharomyces pombe.

DNA replication and DNA repair are essential cell cycle steps ensuring correct transmission of the genome. The feedback replication control system links mitosis to completion of DNA replication and partially overlaps the radiation checkpoint control. Deletion of the chk1/rad27 gene abolishes the radiation but not the replication feedback control. Thermosensitive mutations in the DNA polymerase delta, cdc18 or cdc20 genes lead cells to arrest in the S phase of the cell cycle. We show that strains carrying any of these mutations enter lethal mitosis in the absence of the radiation checkpoint chk1/rad27. We interpret these data as an indication that an assembled replisome is essential for replication dependent control of mitosis and we propose that the arrest of the cell cycle in the thermosensitive mutants is due to the chk1+/rad27+ pathway, which monitors directly DNA for signs of damage.

Cdc20 Proteins↗
PubMed1987

Effects of modulation of RNase H production on the recovery of DNA synthesis following UV-irradiation in Escherichia coli.

The requirements for the recovery of DNA synthesis in UV-irradiated Escherichia coli were analysed in strains having varied levels of RNase H and RecA protein. We have previously shown (Khidhir et al. 1985) that the recovery of DNA synthesis in E. coli following UV treatment is an inducible SOS function requiring protein synthesis. We proposed that this reflected the need for the synthesis of specific induced replisome reactivation factor(s) for recovery. In this study we now show that recovery of DNA synthesis can in fact take place in the absence of protein synthesis in a mutant lacking RNase H and having high (constitutive) levels of RecA protein. We also show that expression of rnh is inhibited during the SOS response in recA+ but not in a recA- strain. The results are discussed in relation to the mechanism of recovery of DNA synthesis following UV irradiation in E. coli.

DNA Repair↗