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At least 1,567 records · Page 87Linked to original sources
PubMed1998

A fifteen-amino-acid peptide inhibits human papillomavirus E1-E2 interaction and human papillomavirus DNA replication in vitro.

Mutation of the conserved glutamic acid residue at position 39 of human papillomavirus type 16 (HPV-16) E2 to alanine (E39A) disrupts its E1 interaction activity and its replication function in transient replication assays but does not affect E2 transcriptional activation. This E39A mutation also disrupts replication activity of HPV-16 E2 in HPV-16 in vitro DNA replication. On this basis, we designed 23- and 15-amino-acid peptides derived from HPV-16 E2 sequences flanking the E39 residue and tested the ability of these peptides to inhibit interaction between HPV-16 E1 and E2 in vitro. The inhibitory activity of these peptides was specific, since analogous peptides in which alanine was substituted for the E39 residue did not inhibit interaction. The 15-amino-acid peptide E2N-WP15 was the smallest peptide tested that effectively inhibited HPV-16 E1-E2 interaction. This peptide also inhibited in vitro replication of HPV-16 DNA. The efficacy of E2N-WP15 was not exclusive to HPV-16: this peptide also inhibited interaction of HPV-11 E1 with the E2 proteins of both HPV-11 and HPV-16 and inhibited in vitro replication with these same combinations of E1 and E2 proteins. These results provide further evidence that E1-E2 interaction is required for papillomavirus DNA replication and constitute the first demonstration that inhibition of this interaction is sufficient to prevent HPV DNA replication in vitro.

Amino Acid Sequence↗
PubMed2000

Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle.

Cdc6 is essential for eukaryotic DNA replication. We have mutated highly conserved CDK phosphorylation sites in Cdc6. Contrary to their reported phenotypes in human cells, unphosphorylatable DeltaCDK mutants fully support DNA replication in Xenopus eggs. WtCdc6 is actively exported from the nucleus, which could explain why nuclear permeabilization is required for reinitiation within one cell cycle. However, DeltaCDK mutants are retained in the nucleus, yet surprisingly they still support only one round of replication. As these highly conserved CDK sites are unnecessary for replication once per cell cycle, an alternative checkpoint role for monitoring completion of the S phase is suggested.

Active Transport, Cell Nucleus↗
PubMed1988

Interaction between the octamer-binding protein nuclear factor III and the adenovirus origin of DNA replication.

Nuclear factor III (NFIII) is a HeLa sequence-specific DNA-binding protein that stimulates initiation of adenovirus DNA replication in vitro and may be involved in regulation of transcription of several cellular and viral genes. We have studied the interaction between NFIII and the binding site in the adenovirus type 2 (Ad2) origin in detail by methidiumpropyl-EDTA.iron(II) and hydroxyl radical footprinting and by alkylation interference experiments. Our results indicate that (i) the core of the recognition sequence is 5'-TATGATAAT-3'; (ii) both major and minor groove base contacts are detected, and all base pairs in the core are involved in binding; (iii) many backbone contacts are observed divided into a large domain coinciding with the core and a small domain; (iv) contact points are not confined to one side of the DNA helix in contrast to the nuclear factor I (NFI)-binding site; (v) the binding site overlaps the NFI-binding site for at least one nucleotide. A number of Ad2 mutants as well as related binding sites in the origins of other adenovirus serotypes were systematically compared for binding with NFIII. The results are in good agreement with the contact point studies and show that at least one AT base pair is commonly required by NFI and NFIII for optimal binding. The strongest binding site, which contains the octamer/decanucleotide motif (ATGCAAAT[NA]), was found in the Ad4 origin, which lacks an NFI-binding site. Stimulation of in vitro DNA replication of Ad2, Ad4, and Ad12 by NFIII showed that the maximal level of stimulation is dependent on the affinity of NFIII for the origin.

Adenoviruses, Human↗
PubMed2006

A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene.

We confirmed that the replication of the fragile-X E site (FRAXE) in human chromosomal band Xq28 occurs at six hours into the eight-hour S phase of normal human fibroblasts. In this late-replicating region, we mapped an origin of DNA replication within the promoter of FMR2. This origin is coincident with CpG islands, the trinucleotide repeat, and exon 1 of the FMR2 gene. Identification of this origin may aid in the investigation of the mechanism of trinucleotide repeat expansion and its effect on FMR2 expression. In addition, knowledge of the chromosomal locations and sequence characteristics of both early and late origins of DNA replication, such as the one described in this report, will facilitate studies of the molecular determinants of the time of activation of different origins of replication and allow us to refine our insights concerning origin inactivation in response to the DNA damage-induced intra-S checkpoint.

Cell Line↗
PubMed2002

Sequence-independent DNA binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

The DnaA protein specifically binds to the origin of chromosomal DNA replication and initiates DNA synthesis. In addition to this sequence-specific DNA binding, DnaA protein binds to DNA in a sequence-independent manner. We here compared the two DNA binding activities. Binding of ATP and ADP to DnaA inhibited the sequence-independent DNA binding, but not sequence-specific binding. Sequence-independent DNA binding, but not sequence-specific binding, required incubation at high temperatures. Mutations in the C-terminal domain affected the sequence-independent DNA binding activity less drastically than they did the sequence-specific binding. On the other hand, the mutant DnaA433, which has mutations in a membrane-binding domain (K327 to I344) was inert for sequence-independent binding, but could bind specifically to DNA. These results suggest that the two DNA binding activities involve different domains and perform different functions from each other in Escherichia coli cells.

Adenine Nucleotides↗
PubMed1982

Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells.

The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with 3H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA.

Anthracenes↗
PubMed2002

Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system.

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5' end and displacing the other strand while progressing in a 3'-to-5' direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5' end of the nick in the process and providing a 3' OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Animals↗
PubMed1998

Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.

The clinical and pathological features of carcinomas of the pancreas with DNA replication errors (RER+) have not been characterized. Eighty-two xenografted carcinomas of the pancreas were screened for DNA replication errors using polymerase chain reaction amplification of microsatellite markers. Cases with microsatellite instability in at least two markers of a minimum of five tested were considered RER+. RER status was correlated with histological appearance, karyotype of the carcinomas when available, K-ras mutational status, and patient outcome. Three (3.7%) of the eighty-two carcinomas were RER+. In contrast to typical gland-forming adenocarcinomas of the pancreas, all three RER+ carcinomas were poorly differentiated and had expanding borders and a prominent syncytial growth pattern. Neither a Crohn's-like lymphoid infiltrate nor extracellular mucin production were prominent. Ductal adenocarcinomas of the pancreas typically contain a mutant K-ras gene, yet all three RER+ carcinomas had wild-type K-ras. One of the three RER+ carcinomas was karyotyped and showed a near diploid pattern. All three of the RER+ tumors were removed via Whipple resection. One of the three patients is free of disease 16 months after pancreaticoduodenectomy, one is alive and free of tumor at 52 months but developed two colon carcinomas during this period, and the third died of pancreatic cancer at 4 months. None of the three patients had a family history of colorectal carcinoma. A review of the K-ras wild-type carcinomas in a previously characterized series of pancreatic carcinomas with known K-ras mutational status identified two additional cancers with poor differentiation, a syncytial growth pattern, and pushing borders. Both of the cancers were diploid and both patients were longterm survivors (over 5 years). The inclusion of such patients in previous prognostic studies of pancreas cancer may explain the failure of histological grade to be a predictor of prognosis. These data suggest that DNA replication errors occur in a small percentage of resected carcinomas of the pancreas and that wild-type K-ras gene status and a medullary phenotype characterized by poor differentiation, and expanding pattern of invasion, and syncytial growth should suggest the possibility of DNA replication errors in carcinomas of the pancreas.

Adenocarcinoma↗
PubMedDate not supplied

DNA replication and indirect induction of the SOS response in Escherichia coli.

The SOS response can be induced indirectly in Escherichia coli by infection with UV irradiated bacteriophage P1, lambda or M13. Induction, monitored quantitatively by means of a sfiA::lac operon fusion, was stronger with the plasmid phage P1 than with lambda, but the kinetics were similar, showing that plasmid and non-plasmid phages are not fundamentally different in their ability to produce indirect induction. In the absence of lambda DNA replication the level of induction was strongly reduced, indicating that the attempt to replicate damaged DNA results in induction of the SOS response. The slight residual induction observed in the absence of DNA replication suggests the existence of a second pathway leading from DNA lesions to induction of the SOS response.

Coliphages↗
PubMed2000

Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts.

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.

Animals↗
PubMed2004

Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site.

Abasic sites are common DNA lesions, which are strong blocks to replicative polymerases and are potentially mutagenic when bypassed. We report here the 2.8 A structure of the bacteriophage RB69 replicative DNA polymerase attempting to process an abasic site analog. Four different complexes were captured in the crystal asymmetric unit: two have DNA in the polymerase active site whereas the other two molecules are in the exonuclease mode. When compared to complexes with undamaged DNA, the DNA surrounding the abasic site reveals distinct changes suggesting why the lesion is so poorly bypassed: the DNA in the polymerase active site has not translocated and is therefore stalled, precluding extension. All four molecules exhibit conformations that differ from the previously published structures. The polymerase incorporates dAMP across the lesion under crystallization conditions, indicating that the different conformations observed in the crystal may be part of the active site switching reaction pathway.

Binding Sites↗
PubMed2004

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D.

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3'-5' proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3'-5' exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.

Amino Acid Sequence↗
PubMed2001

JC virus T' proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells.

Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T'(135), T'(136), and T'(165), were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T' proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T' mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T' proteins have on the replication process, a set of T' acceptor site mutants was created in which the unique second acceptor splice site of each T' mRNA was altered to eliminate production of one, two or all three T' mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T'(135) acceptor site resulted in the utilization of cryptic splice sites and the generation of new T' species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T' protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T' proteins and the double mutant that only produces T'(136), exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T'(136) replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T' donor site mutant from an earlier study, was impaired significantly.

Alternative Splicing↗
PubMed2002

Initiation of DNA replication in eukaryotes is an intriguing cascade of protein interactions.

Initiation of eukaryotic DNA replication is a complex process including the recognition of initiation sites on DNA, multi-step DNA preparation for duplication, and assembly of multi-protein complexes capable of beginning DNA synthesis at initiation sites. The process starts at the late M phase and lasts till the appropriate time of the S phase for each initiation site. A chain of interesting interactions between Orc1p-6p, Cdc6p, Mcm2p-7p, Mcm10p, Cdt1, Cdc45p, Dbf4/Cdc7p, RPA, and DNA polymerase alpha takes place during this period. The sequence of these interactions is controlled by cyclin-dependent kinases, as well as by ubiquitin-dependent proteolysis in the proteasome. This review summarizes the data on proteins initiating DNA replication and factors controlling their activities.

Binding Sites↗
PubMed2005

Coordination of DNA replication and cell division in cyanobacteria Microcystis aeruginosa.

Little is known about the cyanobacterial cell cycle. When either nalidixic acid or hydroxyurea was added to a synchronized culture of Microcystis aeruginosa to block DNA replication, cell division did not occur. Furthermore, transcription of the essential cell division gene, ftsZ was repressed. After DNA replication, ftsZ transcription, as well as cell division, was not affected by hydroxyl urea, suggesting that the DNA replication and cell division of M. aeruginosa are coordinated and that this coordination is partly controlled by ftsZ transcription depending on DNA replication.

Bacterial Proteins↗
PubMed1998

Initiation of DNA replication in phages and plasmids-a workshop summary.

Recent progress in understanding initiation mechanisms of DNA replication was discussed by some 30 speakers from Europe and U.S.A. in a workshop funded entirely by the Juan March Foundation of Madrid. Several speakers were postdoctoral associates and were selected from poster presenters. This was probably the first meeting at which phage and plasmid systems were given top billing together. The confluence was highly successful because of considerable overlap in strategies employed by the two systems. Mechanistic studies on DNA replication started with phage and the lessons learned have guided the thinking on eukaryotic DNA replication (Stillman, J. Biol. Chem. (1994) 269, 7047). The research on bacterial plasmids has also been pioneering in defining the principles of initiation control and has unraveled novel biological regulatory mechanisms such as antisense control.

Bacteriophages↗
PubMed1976

Nucleocytoplasmic ratio requirements for the initiation of DNA replication and fission in Tetrahymena.

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S-phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. 3H-TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G1. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2-4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post-fission-daughter cells and pre-fission cells is identical (standardized to N/C = 1) but late G1 cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4X and the C continues to increase until just prior to recovery division when it also reaches a value 4X that of the original daughter cells. Thus, the N/C = 1 is re-established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G1. The elimination of G1 (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.

Animals↗
PubMed1989

Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication.

We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near ori lambda and proceeds unidirectionally rightwards through theta-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of lambda DNA replication in the purified protein system does not require "transcriptional activation" of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system.

Bacterial Proteins↗