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At least 1,099 records · Page 61Linked to original sources
PubMed2003

Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis.

In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end. DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.

Bacillus subtilis↗
PubMedDate not supplied

[Enzymatic mechanisms of degradation of DNA replication forks in vitro].

The present work provides experimental evidence of two ways of fermentative degradation of DNA replication forks in vivo. The first way proceeds under the influence of exonuclease V, which degradates DNA replication forks, uncovered by singlestrand binding (ssb) proteins. DNA replication forks protected against nucleases by ssb protein are degrated by a second way: by tandem action of DNA polymerase II with ssb protein complexes and exonuclease V. The default of normal ssb protein in cells of Escherichia coli is responsible for the first way of DNA replication forks degradation. The second way is occurs at excess of normal ssb protein.

Bacterial Proteins↗
PubMed1991

Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells.

In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to [3H]thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

Caffeine↗
PubMed2002

Xenopus Cdc6 performs separate functions in initiating DNA replication.

Cdc6 performs an essential role in the initiation of eukaryotic DNA replication by recruiting the minichromosome maintenance (MCM) complex onto DNA. Using immunodepletion/add-back experiments in Xenopus egg extracts, we have determined that both Walker A (ATP binding) and Walker B (ATP hydrolysis) motifs of Xenopus Cdc6 (Xcdc6) are essential, but have distinct functional roles. Although Walker B mutant protein binds chromatin well, Walker A mutant protein binds chromatin poorly. Neither Walker A nor Walker B mutant protein, however, load appreciable MCM onto DNA. Herein, we provide evidence that Cdc6 functions as a multimer: 1) mutant and wild-type Xcdc6 form multimers; 2) either mutant protein is dominant negative when added before wild-type Xcdc6, but stimulates DNA replication when added simultaneously with wild-type Xcdc6; and 3) the two mutants restore DNA replication when added together, in the absence of wild-type Xcdc6. Our findings suggest that ATP may play a key regulatory role within this multimer: its binding to Cdc6 promotes chromatin association and its hydrolysis facilitates MCM loading. Moreover, ATP binding and hydrolysis may occur in trans between Cdc6 subunits within the complex.

Adenosine Triphosphatases↗
PubMed1983

Immunological characterization of the role of adenovirus terminal protein in viral DNA replication.

The function of the adenovirus-coded terminal protein and its precursor in viral DNA replication was studied by raising an antiserum against the adenovirus type 5 (Ad5) terminal protein isolated from virions. This antiserum reacted with both the terminal protein and its precursor as measured by a radioimmunoassay. In an in vitro DNA replication system employing nuclear extracts the addition of antiserum inhibits replication when a DNA-terminal protein complex from adenovirions is used as template. The replication of a 3.8% terminal fragment of the Ad2 genome with a protein-free origin (derived from the plasmid XD-7) is also inhibited by the antiserum. This observation confirms a role of the terminal protein precursor in DNA replication. The antiserum completely inhibited the formation of a covalent complex between the precursor terminal protein and dCMP, which is essential for initiation. A function of the terminal protein in the elongation reaction was shown by the inhibitory effect of antiserum on DNA chain elongation in isolated nuclei from Ad5-infected cells. Also in the in vitro DNA replication system employing nuclear extracts the elongation reaction is strongly reduced by addition of the antiserum. These results indicate that the terminal protein and/or its precursor are not only involved in initiation of DNA replication but also in DNA chain elongation.

Adenovirus Infections, Human↗
PubMed1999

Identification of initiation sites for DNA replication in the human dnmt1 (DNA-methyltransferase) locus.

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information. Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that the dnmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

Chromosome Mapping↗
PubMed1996

cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication.

A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termed ROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM3 subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2-4 copies per haploid genome of closely related ROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels of ROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript. In situ hybridisation analysis in the root apex reveals that ROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley and Arabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.

Amino Acid Sequence↗
PubMed2001

Replisome-mediated DNA replication.

The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

Bacterial Proteins↗
PubMed2003

Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication.

Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three-dimensional structure of the full-length T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single-particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer-hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.

Algorithms↗
PubMed2004

Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation.

DNA damage-induced S phase (S) checkpoint includes inhibition of both replicon initiation and chain elongation. The precise mechanism for controlling the two processes remains unclear. In this study, we showed that Hus1-deficient mouse cells had an impaired S checkpoint after exposure to DNA strand break-inducing agents such as camptothecin (CPT) (>or=1.0 micro M), or ionizing radiation (IR) (>or=15 Gy). The Hus1-dependent S checkpoint contributes to cell resistance to CPT. This impaired S checkpoint induced by CPT or IR in Hus1-deficient cells reflected mainly the chain elongation step of DNA replication and was correlated with the reduction of dissociation of PCNA from DNA replication foci. Although Hus1 is required for Rad9 phosphorylation following exposure of cells to CPT or IR, Hus1-deficient cells showed normal activation of ATR/CHK1 and ATM kinases at doses where the checkpoint defects were manifested, suggesting that Hus1 is not a component of the sensor system for activating these pathways in S checkpoint induced by CPT or IR.

Animals↗
PubMed1989

Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo.

Initiation of simian virus 40 (SV40) DNA replication is facilitated by two auxiliary sequences that flank the minimally required origin (ori) core sequence. In monkey cells, the replication rate of each of the four ori configurations changed with time after transfection in a characteristic pattern. This pattern was reproduced in an extract from SV40-infected monkey cells by varying the ratio of DNA substrate to cell extract; DNA replication in vitro depended on ori auxiliary sequences to the same extent as they did in vivo. Facilitation by ori auxiliary sequences was lost at high ratios of DNA to cell extract, revealing that the activity of these sequences required either multiple initiation factors or a molar excess of one initiation factor bound to ori. This parameter, together with ionic strength and the method used to measure DNA replication, determined the level of facilitation by ori auxiliary sequences in vitro. The activity of ori auxiliary sequences was not diminished in vivo or in vitro by increasing amounts of large tumor antigen. Therefore, ori auxiliary sequences promoted initiation of replication at some step after tumor antigen binding to ori. Furthermore, although cellular factors could modulate the activity of ori auxiliary sequences in vitro, these factors did not appear to involve nucleosome assembly because no correlation was observed between the number of nucleosomes assembled per DNA molecule and facilitation by ori auxiliary sequences. These results demonstrate that SV40 ori auxiliary sequences can function in vitro as they do in vivo and begin to elucidate their role in initiating DNA replication.

Animals↗
PubMed1981

Cell density dependent DNA replication in Ehrlich ascites tumour cells.

DNA replication of Ehrlich ascites tumour cells was investigated in suspensions with different cell densities by incorporation in vitro of tritiated thymidine and alkaline sucrose gradient analysis of the newly formed DNA. It is demonstrated that the incorporation of [3H]thymidine and chain growth of newly made DNA decreases with increasing cell density. The inhibition of DNA synthesis observed at high cell densities can be prevented if diffusible substances are removed by incubating the cells in dialysis tubes. This indicates that the changes in DNA synthesis are caused by diffusible inhibitors released from the tumour cells.

Animals↗
PubMed1983

Effect of aphidicolin on the elongation step of adenovirus DNA replication in vitro.

Adenovirus DNA synthesis carried out in vitro was inhibited by the aphidicolin. However, 30% of the DNA synthesis was resistant to aphidicolin even at a concentration of 200 micrograms/ml. When the distribution patterns of the radioactivity of the products synthesized in the presence of 50 micrograms/ml of the drug was examined after HindIII digestion of the product DNA, the radioactivity appeared preferentially in the fragments mapping nearest to the ends of the molecule. Pulse-chase experiment showed that the terminal fragments were synthesized with or without aphidicolin but that in the presence of aphidicolin the rate of elongation rapidly slowed down beyond this region, suggesting that a DNA polymerase sensitive to aphidicolin may participate in the synthesis of the internal region of adenovirus DNA.

Adenoviridae↗
PubMed1996

Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp.

The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA.

Adenosine Triphosphate↗
PubMed1975

Isolation of DNA polymerase gamma from an adenovirus 2 DNA replication complex.

The major DNA polymerase in a nuclear membrane complex that is capable of synthesizing viral DNA sequences in vitro has been purified about 900-fold from adenovirus 2-infected KB cells. The enzyme was characterized as belonging to the class of mammalian DNA polymerases (DNA polymerase gamma) that can utilize poly(A) with oligo(dT) as template primer.

Adenoviridae↗
PubMedDate not supplied

[Role of glucocorticoids in the mitochondrial DNA replication].

The liver mitochondrial DNA biosynthesis was found to be enhanced in adrenalectomized rats. Cortisol administration to adrenalectomized rats caused a decrease of the liver mitochondrial DNA biosynthesis. Sedimentation analysis of the mitochondrial DNA in CsCl--EtBr gradient has demonstrated that 3H-thymidine incorporation into the mictochondrial DNA increased mainly in the "open circular" molecules fraction.

Adrenalectomy↗