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At least 289 records · Page 16Linked to original sources
PubMed1995

Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication.

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.

DNA Helicases↗
PubMed2004

Cytotoxicity of psammaplin A from a two-sponge association may correlate with the inhibition of DNA replication.

BACKGROUND: SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied. METHODS: Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase alpha-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA). RESULTS: We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase alpha-primase is one of its main targets. CONCLUSION: Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.

Animals↗
PubMed2005

Replication of simian virus 40 (SV40) DNA in virus-infected CV1 cells selectively permeabilized for small molecules by Staphylococcus aureus alpha-toxin: involvement of mitochondria in the fast O2-dependent regulation of SV40 DNA replication.

SV40 (simian virus 40)-infected CV1 cells were permeabilized with Staphylococcus aureus alpha-toxin for small molecules (<2 kDa) in a medium that supports DNA replication. Incorporation of [alpha-32P]dATP was shown to proceed at an essentially constant rate for at least 1 h. 32P-labelled DNA replication intermediates and products were analysed by alkaline sucrose density centrifugation. The results suggested that SV40 DNA replication in alpha-toxin-permeabilized CV1 cells occurred essentially as in vivo. After bromodeoxyuridine 5'-triphosphate-labelling and isopycnic banding, significant amounts of DNA density-labelled in both strands were detected from 110 min of permeabilization onwards, indicating repeated rounds of viral DNA replication in the permeabilized cells. Incubation of permeabilized SV40-infected cells under hypoxic culture conditions caused inhibition of SV40 DNA replication. As seen in unpermeabilized cells, SV40 DNA replication was inhibited at the stage of initiation. The inhibition of DNA replication induced by hypoxia was mimicked by AA (antimycin A), an inhibitor of mitochondrial respiration, and also by the replacement of glutamate, a substrate of mitochondrial respiration, by Hepes in the permeabilization medium. Inhibition of DNA replication was not mediated by intracellular ATP depletion. AA also inhibited SV40 DNA replication in unpermeabilized, normoxically incubated cells. Moreover, as in hypoxically incubated cells, the addition of glucose to SV40-infected cells incubated for several hours with AA induced a burst of new initiations followed by a nearly synchronous round of viral DNA replication. Taken together, these results indicate that mitochondria are involved in the oxygen-dependent regulation of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed1973

Multiple and specific initiation of T4 DNA replication.

Partially replicated T4 DNA molecules (PRM) whose parental or progeny DNA was labeled with bromodeoxyuridine BUdR was analyzed by gradual shearing followed by CsCl banding of the sheared product. Analysis of PRM containing 18-mum replicated DNA showed that each replicated region was 3- to 6-mum long, indicating three to 6 replicative sites per molecule. Analysis of PRM containing 9-mum replicated DNA similarly indicated two to three replicated regions per molecule. DNA from the replicated regions of PRM containing 10-mum replicated DNA ("donor") was hybridized to DNA from mature phage ("recipient"), and the resulting hybrid was subjected to digestion with exonuclease I. The extent of protection of the recipient and more efficient self-annealing of progeny fragments from PRM indicated that the replicated regions represented 8 to 10 nonrandom locations of the genome. Possible significance of multiple sites for initiation of DNA replication is discussed.

Bromodeoxyuridine↗
PubMed2004

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication.

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.

Antibodies, Monoclonal↗
PubMed2001

Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint.

The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks. It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place. Mec1 and Rad53 also slow S phase progression in response to DNA alkylation, although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1. The accelerated S phase in checkpoint mutants, therefore, is primarily a consequence of inappropriate initiation events. Wild-type cells ultimately complete DNA replication in the presence of MMS. In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates. Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage. Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation.

Alkylating Agents↗
PubMed2000

PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates microreview.

The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D-loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3' to 5' helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA's repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.

Bacteriophage T4↗
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↗
PubMed1992

Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.

Bacillus Phages↗
PubMed2006

Characterization of the uracil-DNA glycosylase activity of Epstein-Barr virus BKRF3 and its role in lytic viral DNA replication.

Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediate-early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

Cells, Cultured↗
PubMed1981

Evidence for double replication of chromosomal DNA segments as a general consequence of DNA replication inhibition.

We have previously presented evidence that a transient inhibition of DNA synthesis by a pulse of 1-beta-D-arabinofuranosylcytosine (ara-C) results in a disruption of the pattern of replication of the chromosomal DNA of cultured human cells, resulting in some DNA segments being replicated more than once in a single S phase. Further evidence is presented in this paper that this effect is not a specific property of the ara-C molecule in that a similar effect is produced in cells by a pulse of 9-beta-D-arabinofuranosyladenine (ara-A) and also by a pulse of cycloheximide. The activated form of ara-A and ara-C (the triphosphates) both inhibit DNA synthesis at the level of the polymerase. Double replication following an ara-A pulse demonstrates that double replication after an ara-C pulse is not caused by some specific property of the ara-C molecule which might be unrelated to any effect on DNA synthesis. However, cycloheximide is an inhibitor of mammalian protein synthesis and inhibits DNA synthesis only indirectly, probably through a consequent deficiency of DNA-packaging proteins. Hence, the occurrence of double replication of chromosomal DNA segments following a pulse of cycloheximide is consistent with this phenomenon being a general and nonspecific consequence of the freezing of DNA replication forks.

Cell Line↗
PubMed1987

2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta.

It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.

Cell Line↗
PubMed1997

Phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases regulates initiation of DNA replication in vitro.

DNA polymerase alpha-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase alpha-primase. The p180 and the p68 subunits of DNA polymerase alpha-primase were phosphorylated using Cyclin A-, B- and E- dependent kinases. This phosphorylation did not influence its DNA polymerase activity on activated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of primers. In contrast, site-specific initiation of replication on plasmid DNA containing the SV40 origin is affected: Cyclin A-Cdk2 and Cyclin A-Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B-Cdc2 had no effect and Cyclin E-Cdk2 stimulated the initiation reaction. DNA polymerase alpha-primase that was pre-phosphorylated by Cyclin A-Cdk2 was completely unable to initiate the SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase alpha-primase remained fully active in the initiation reaction.

CDC2-CDC28 Kinases↗
PubMed1999

Geminivirus DNA replication.

Geminiviruses are DNA viruses which infect plants. They have a small genome and encode only a few proteins. Therefore, their DNA replication cycle relies largely on the use of cellular DNA replication proteins. The strategy used by geminiviruses to replicate their single-stranded DNA (ssDNA) genome consists of a first stage of conversion of ssDNA into double-stranded DNA (dsDNA) intermediates and, then, the use of dsDNA as a template to amplify viral dsDNA and to produce mature ssDNA genomes by a rolling-circle replication mechanism. In addition, the accumulating evidence indicates that viral DNA replication is somehow coupled to the cell cycle regulatory network of the infected cell. For these reasons, geminiviruses are excellent model systems to understand the regulation of DNA replication and cell cycle in plant cells. Recent years have witnessed significant progress in the identification of cis-acting signals and their interaction with trans-acting factors that contribute to geminivirus origin function. These and other aspects of the geminivirus DNA replication cycle will be reviewed.

DNA Replication↗
PubMed1982

DNA replication in Physarum polycephalum: electron microscopic analysis of patterns of DNA replication in the presence of cycloheximide.

DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibitor cycloheximide. The replication-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of replication-loop frequency showed a similar pattern in control and cyclo-heximide-treated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that initiations of new replicons either may not be inhibited by cycloheximide or, alternatively, that all initiations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here.

Cycloheximide↗
PubMed2001

Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication.

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3'-5' exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.

Animals↗
PubMed1991

The nuclear protooncogenes c-jun and c-fos as regulators of DNA replication.

Polyomavirus (Py) DNA replication is regulated by its enhancer, which contains an AP1 site, c-Jun and c-Fos, the products of nuclear protooncogenes c-jun and c-fos, form the heterodimeric transcriptional activating factor AP1. Overexpression of c-fos and c-jun genes strongly stimulated Py DNA replication from the Py origin of replication as well as transcription from the Py early promoter through the AP1 binding site. The cAMP response element (CRE)-binding protein CREB stimulated only transcription, not DNA replication, through the CRE under similar conditions. The results indicate that AP1 functions as a regulator of DNA replication and that the mechanism of activation of Py DNA replication by AP1 is distinct from that of activation of transcription from the Py early promoter.

Animals↗