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At least 1,135 records · Page 63Linked to original sources
PubMed1993

DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites.

We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5' upstream regions of some histone genes.

Animals↗
PubMed1993

Two early replicated, developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis.

The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells. We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase. A single, discrete origin of replication was found within 4.8 kb of the LAV1-5 gene, which encodes a homolog of profilin. In contrast, the LAV1-2 gene appears to be surrounded by several origins. Two origins were identified within a 15 kb chromosomal domain and appear to be inefficiently used. Replication forks collide at preferred sites within this domain. These terminating structures are long lived, persisting for at least 2 h of the 3 h S-phase. Analysis of restriction fragment length polymorphisms (RFLPs) within the LAV1-2 domain indicates that replication of alleles on different parental chromosomes is a highly coordinated process. Our studies of the these two early replicated, plasmodium-specific genes indicate that both a fixed, narrow origin region and a broader zone containing two closely spaced origins of DNA replication occur in Physarum.

Animals↗
PubMed1964

MAMMALIAN CHROMOSOMES IN VITRO. 18. DNA REPLICATION IN THE CHINESE HAMSTER.

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H(3)-thymidine labeling and the method of 5-fluorodeoxyuridine block with H(3)-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.

Animals↗
PubMed1992

Temporal expression of a membrane-associated protein putatively involved in repression of initiation of DNA replication in Bacillus subtilis.

A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990).

Acetyltransferases↗
PubMed1980

Effects of 2',3'-dideoxythymidine triphosphate on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells.

2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled DNA synthesis.

Animals↗
PubMed1983

DNA replication by novel macromolecular complexes involving DNA polymerase III holoenzyme activity.

A fraction (P1) which showed DNA polymerase III holoenzyme activity was obtained by partial purification including Polymin P fractionation from extracts of E. coli K-12 wild type (pol A+, pol B+) cells. The P1 fraction was composed of three macromolecular complexes, 11 S, 18 S and 24 S, all of which possessed holoenzyme activity. The activity of the P1 fraction was maximal at about 70 mM NaCl. The synthesis of long-chain poly(dT) with a poly(dA) oligo(dT)10 primer was dependent on the presence of ATP, but not on the presence of spermidine, suggesting that the single-stranded DNA binding protein (SSB) was present in the fraction. The intermediate lengths of the products in the absence of ATP and NaCl also suggest the functioning of DNA polymerase III'.

Adenosine Triphosphate↗
PubMed1984

On the fidelity of DNA replication. The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro.

The fidelity with which wild type T4 DNA polymerase copies phi X174 amber 3 plus strand DNA at position 587 in vitro has been measured. Synthesis is initiated by hybridizing to the template a HaeIII restriction fragment whose 3'-OH terminus is 83 nucleotides from the amber 3 site. Based on gel electrophoresis of product DNA molecules and genetic marker rescue data, T4 DNA polymerase copies significantly beyond the mutant site. Transfection analysis shows that the A X T leads to G X C mutation at position 587 occurs 10- to 100-fold less frequently with T4 DNA polymerase than with E. coli DNA polymerase I. The aberrant incorporation of cytosine opposite adenine at position 587 by the T4 polymerase alone is occurring at a frequency not greater than about 10(-7) which, for this particular locus, may be similar to the fidelity exhibited by the T4 accessory proteins plus the polymerase comprising the replication complex. A comparison of the accuracy of mutator L56 and antimutator L141 T4 DNA polymerases relative to wild type shows at most a 2- to 4-fold decrease and increase, respectively, in fidelity. When compared to 10- to 1000-fold effects on mutation frequencies that these same mutant alleles have in vivo, these results suggest that the wide range in expression of mutator and antimutator phenotypes in vivo may be dependent on an abnormal interaction of the aberrant DNA polymerases with other protein components of the replication complex.

Bacteriophage phi X 174↗
PubMed1985

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.

Animals↗
PubMed1997

An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha.

Epstein-Barr virus (EBV) replicates as a stable multicopy episome in latently infected mammalian cells. Latent cycle DNA replication requires only two viral elements, the cis-acting origin of plasmid replication (oriP) and the trans-acting origin binding protein (EBNA1). EBNA1 binds multiple recognition sites in oriP, but has not other enzymatic activities associated with replication functions. To identify human cellular proteins that mediate EBNA1 function, we designed a one-hybrid assay in yeast to select for proteins that bind to EBNA1 when bound to criP in vivo. A human cDNA encoding the Rch1/hSRP1 alpha/ importin alpha protein was isolated and shown to bind to full-length EBNA1, but not to an amino terminal deletion mutant of EBNA1 when bound to oriP in yeast. The interaction of EBNA1 with Rch1 was confirmed biochemically by coimmunoprecipitation from nuclear extracts and by direct binding of recombinant proteins in vitro. Internal deletion mutations in EBNA1 which compromised DNA replication activity were similarly reduced for binding to Rch1. Mutations with no effect on DNA replication activity were similarly unaffected for Rch1 binding. Rch1/importin alpha has been shown to bind to the nuclear localization sequence (NLS) of several proteins and stimulate nuclear import. A substitution mutation in the EBNA1 nuclear localization sequence reduced Rch1 binding, but had no effect on DNA replication function, indicating that Rch1 binding affinity does not correspond precisely with replication activity. Nevertheless, the identification of a stable interaction between Rch1 and EBNA1 at the origin of viral DNA replication raises the intriguing possibility that Rch1 contributes to the nuclear functions of EBNA1.

Amino Acid Sequence↗
PubMed2001

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

Amino Acid Motifs↗
PubMed2001

DNA replication. Genomic views of genome duplication.

DNA replication is initiated at numerous origins of replication (oris) within the chromosomes. In a pair of ambitious studies, two groups have used different techniques to pinpoint the locations of all of the oris throughout the yeast genome at different times during S phase (Raghuraman et al., Wyrick et al.). Stillman, in his Perspective, compares and contrasts the different methods and their findings, and speculates on the value of combining these techniques to look at oris in the human genome.

Binding Sites↗
PubMed1989

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates.

Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein). Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded. However, the other pause sites are in regions that are not obviously involved in secondary structure. The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides. At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions. The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing. In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing. This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.

Base Sequence↗
PubMed1998

The UL112/113 gene products of human cytomegalovirus which colocalize with viral DNA in infected cell nuclei are related to efficient viral DNA replication.

The UL112/113 gene products of human cytomegalovirus (HCMV) were shown by transient complementation ori Lyt-dependent DNA replication assay to be early viral proteins required for efficient viral DNA synthesis. By immunofluorescence analysis followed by fluorescence in situ hybridization, we showed that UL112/113 gene products of HCMV are colocalized with viral DNA prior to and during viral DNA replication in infected cell nuclei. We have used an anti-sense RNA approach for functional analysis of the UL112/113 gene in HCMV. The astrocytoma cell line U373-MG was used for permanent expression of the anti-sense UL112/113 gene. Expression of the anti-sense RNA in this cell line significantly blocked expression of UL112/113 gene products and viral DNA replication, indicating that the UL112/113 gene products are related to efficient viral DNA replication.

Cell Line↗
PubMed1994

DNA replication of chimeric JC virus-simian virus 40 genomes.

The ubiquitous virus JCV is the etiologic agent of the human brain disease progressive multifocal leukoencephalopathy. Although infection usually occurs early in life and the virus can remain latent in human tissues, including brain, little information is available regarding its replication. It is known that DNA replication of primate polyomaviruses is dependent upon the synthesis of T antigen and the subsequent interactions of this protein with cellular factors and the viral origin of replication. We constructed chimeric genomes between JCV and SV40, two genetically similar viruses with distinct biologies, in which segments of the T antigen coding region and the replication origin were exchanged. Because the engineering of these genomes created a defect in the structural protein VP1, their DNA replicating activities could be compared without the complication of secondary infection of adjacent cells and amplification of the replication signal. The ability of the JCV-SV40 hybrid T antigens to initiate replication from the two viral origins in primate cells was investigated. A region of the JCV T antigen that includes the DNA binding and zinc finger domains was found to be responsible for the failure of JCV T antigen to interact productively with the SV40 origin. In addition, the ability to replicate in monkey cells was limited to constructs expressing T antigens which contained the carboxy-terminal host range domain of SV40.

Antigens, Polyomavirus Transforming↗
PubMed1999

Regulation of eukaryotic DNA replication and nuclear structure.

In eukaryote, nuclear structure is a key component for the functions of eukaryotic cells. More and more evidences show that the nuclear structure plays important role in regulating DNA replication. The nuclear structure provides a physical barrier for the replication licensing, participates in the decision where DNA replication initiates, and organizes replication proteins as replication factory for DNA replication. Through these works, new concepts on the regulation of DNA replication have emerged, which will be discussed in this minireview.

Animals↗
PubMed1970

Chromosomal DNA replication pattern in human tumour cells in vitro.

The present paper deals with the chromosomal DNA replication pattern in human solid tumour cells in vitro. This was studied at the terminal stages of the S-period. All the cell lines of female origin showed a late replicating chromosome in group XX6-12. In cell lines of male origin one of the chromosomes of group 21-22Y was later replicating than the rest of the members of the group. The DNA replication pattern of the autosomes and the sex chromosomes was similar to that of the cultured human leucocytes. The results of the present study show that the DNA replication pattern of the chromosome in neoplastic cells is basically unchanged despite the changes in the chromosome number and morphology. Therefore the abnormal behaviour of the neoplastic cells cannot be related to the changes in the pattern of the chromosomal DNA replication.

Astrocytoma↗