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At least 883 records · Page 49Linked to original sources
PubMed2002

The role of single-stranded DNA and polymerase alpha in establishing the ATR, Hus1 DNA replication checkpoint.

Using a nucleus-free DNA replication system we have investigated the roles of Xenopus ATR (XATR) and Hus1 (Xhus1) as the DNA replication checkpoint sensors. Like XATR, Xhus1 is required for the checkpoint-dependent phosphorylation of Xchk1 and associates with chromatin in an initiation-dependent manner. While removal of replication protein A inhibits chromatin association of both XATR and Xhus1, removal of polymerase alpha only disrupts chromatin association of Xhus1. In addition, chromatin association of XATR and Xhus1 are independent of each other. Finally, like XATR, Xhus1 associates with chromatin in unperturbed S phase and dissociates from chromatin following completion of DNA replication.

Animals↗
PubMed1983

[DNA replication in mammalian cells acted on by chemical, physical and biological factors. I. DNA damage and replication in LL line cells treated with formaldehyde].

The inhibition of DNA synthesis and the appearance of single-strand breaks and/or alkali-labile sites in DNA and DNA-membrane cross-links were observed after formaldehyde treatment of cultured LL-line cells. It was shown that supercoiling of cell chromatin is not affected under these conditions. The initiation of DNA replication after the exposure with 10(-4) M formaldehyde occurs also without disturbance. Under the higher concentration of formaldehyde (10(-3) M), DNA elongation was inhibited. It is suggested that cross-linking of DNA with other molecules and structures for example membranes, stabilizes DNA supercoiling (chromatine). This conformational stability is essential for normal initiation of DNA replication, although the parenteral DNA contains many lesions in its primary and secondary structures.

Animals↗
PubMed1996

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.

Animals↗
PubMed2001

A tale of two HSV-1 helicases: roles of phage and animal virus helicases in DNA replication and recombination.

Helicases play essential roles in many important biological processes such as DNA replication, repair, recombination, transcription, splicing, and translation. Many bacteriophages and plant and animal viruses encode one or more helicases, and these enzymes have been shown to play many roles in their respective viral life cycles. In this review we concentrate primarily on the roles of helicases in DNA replication and recombination with special emphasis on the bacteriophages T4, T7, and A as model systems. We explore comparisons between these model systems and the herpesviruses--primarily herpes simplex virus. Bacteriophage utilize various pathways of recombination-dependent DNA replication during the replication of their genomes. In fact the study of recombination in the phage systems has greatly enhanced our understanding of the importance of recombination in the replication strategies of bacteria, yeast, and higher eukaryotes. The ability to "restart" the replication process after a replication fork has stalled or has become disrupted for other reasons is a critical feature in the replication of all organisms studied. Phage helicases and other recombination proteins play critical roles in the "restart" process. Parallels between DNA replication and recombination in phage and in the herpesviruses is explored. We and others have proposed that recombination plays an important role in the life cycle of the herpesviruses, and in this review, we discuss models for herpes simplex virus type 1 (HSV-1) DNA replication. HSV-1 encodes two helicases. UL9 binds specifically to the origins of replication and is believed to initiate HSV DNA replication by unwinding at the origin; the heterotrimeric helicase-primase complex, encoded by UL5, UL8, and UL52 genes, is believed to unwind duplex viral DNA at replication forks. Structure-function analyses of UL9 and the helicase-primase are discussed with attention to the roles these proteins might play during HSV replication.

Animals↗
PubMed1999

The functional role of a DNA primase in chloroplast DNA replication in Chlamydomonas reinhardtii.

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11-18, 5-12, and 3-11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.

Animals↗
PubMed1992

Cell specificity of transcription regulation by papovavirus T antigens and DNA replication.

Simian virus 40 (SV40) and polyomavirus (Py) DNA replication require cellular proteins and a virus-encoded early gene product, large T antigen (SVT and PyT, respectively). Primate cells contain factors permissive for SV40 replication, whereas murine cells express those factors permissive for Py. We have compared the roles T antigen, cell permissiveness and replication play in transcription of SV40 and Py genes. We show that in their respectively permissive cells, SV40 replication causes a major shift in transcription initiation from the early to the late viral promoter, whereas when Py replicates a comparable shift does not occur. This difference is discussed in relation to differences in the organization of the origin and promoter region between these two papovaviruses. Reporter plasmids were constructed that carried both viral origins, one at the natural position in the promoter being tested and the other at a distal location. With the appropriate TAg, these vectors could be made to replicate in either primate (HeLa) or rodent (3T6) cells. The SV40 early to late shift occurred when replication was driven in HeLa cells, and was not seen on replicating templates in rodent cells. Thus, replication per se does not account for the shift. We show also that, like SVT, PyT is a potent activator of transcription, and that SVT and PyT can activate each other's late promoters independently of DNA replication, but only in cells permissive for DNA replication catalysed by the respective T antigen. Taken together, the data presented here suggest that papovaviruses may utilize permissive factors in transcription control mechanisms.

Animals↗
PubMed1985

Monoclonal antibodies against human DNA polymerase-alpha inhibit DNA replication in permeabilized human cells.

Monoclonal neutralizing antibodies against DNA polymerase-alpha substantially inhibit nuclear DNA replication in lysolecithin-permeabilized cultured human fibroblasts. The degree of inhibition of DNA synthesis is proportional to antibody concentration, and the effect is specific in that RNA synthesis measured under the same experimental conditions is unperturbed. Autoradiographic data demonstrate that the magnitude of the inhibition measured in the mass culture reflects the uniform response of all the constituent cells in the target population. These observations confirm the participation of DNA polymerase-alpha in replicative DNA synthesis and identify a versatile, novel approach to the dissection of mammalian processes of DNA replication and repair.

Antibodies, Monoclonal↗
PubMed1979

Metabolism of Okazaki fragments during simian virus 40 DNA replication.

Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.

DNA Ligases↗
PubMed2004

Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7.

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.

Bacteriophage T7↗
PubMed2002

Initiation of eukaryotic DNA replication: regulation and mechanisms.

The accurate and timely duplication of the genome is a major task for eukaryotic cells. This process requires the cooperation of multiple factors to ensure the stability of the genetic information of each cell. Mutations, rearrangements, or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing failures, disease, or death. Because of the size of eukaryotic genomes, chromosomal duplication is accomplished in a multiparallel process. In human somatic cells between 10,000 and 100,000 parallel synthesis sites are present. This raises fundamental problems for eukaryotic cells to coordinate the start of DNA replication at each origin and to prevent replication of already duplicated DNA regions. Since these general phenomena were recognized in the middle of the 20th century the regulation and mechanisms of the initiation of eukaryotic DNA replication have been intensively investigated. These studies were carried out to find the essential factors involved in the process and to determine their functions during DNA replication. These studies gave rise to a model of the organization and the coordination of DNA replication within the eukaryotic cell. The elegant experiments carried out by Rao and Johnson (1970) (1), who fused cells in different phases of the cell cycle, showed that G1 cells are competent for replication of their chromosomes, but lack a specific diffusible factor required to activate their replicaton machinery and showed that G2 cells are incompetent for DNA replication. These findings suggested that eukaryotic cells exist in two states. In G1 phase, cells are competent to initiate DNA replication, which is subsequently triggered in S phase. After completion of S phase, cells in G2 are no longer able to initiate DNA replication and they require a transition through mitosis to reenable initiation of DNA replication to take place in the next S phase. The Xenopus cell-free replication system has proved a good model system in which to study DNA replication in vitro as well as the mechanism preventing rereplication within a single cell cycle (2). Studies using this system resulted in the development of a model postulating the existence of a replication licensing factor, which binds to chromatin before the G1-S transition and which is displaced during replication (2, 3). These results were supported by genetic and biochemical experiments in Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) (4, 5). The investigation of cell division cycle mutants and the budding yeast origin of replication resulted in the concept of a prereplicative and a postreplicative complex of initiation proteins (6-9). These three individual concepts have recently started to merge and it has become obvious that initiation in eukaryotes is generally governed by the same ubiquitous mechanisms.

Animals↗
PubMed1995

Regulation of DNA replication in irradiated cells by trans-acting factors.

We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 (SV40)-based in vitro replication assay. The assay measures semi-conservative DNA replication in a plasmid carrying the SV40 origin of replication and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radiation. We find that replication of plasmid DNA is significantly reduced when cytoplasmic extracts from irradiated cells are used. Since plasmid replication proceeds to completion in extracts from irradiated cells, the observed reduction in the over-all replication activity is probably due to a reduction in the efficiency of initiation events. The degree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured in vitro using this assay is similar to that measured in intact cells immediately before processing for extract preparation. These observations are compatible with the induction or activation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechanism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the restoration of DNA integrity, and may define yet another checkpoint operating during S at the level of clusters of replicons.

Animals↗
PubMed1983

In vitro complementation as an assay for purification of adenovirus DNA replication proteins.

As an approach to the purification of adenovirus-encoded DNA replication proteins, we have developed in vitro complementation assays that make use of viral mutants defective in DNA replication in vivo. Nuclear extracts prepared from cells infected with H5ts36 or H5ts125, two such mutants belonging to different complementation groups, were found to be defective in viral DNA replication in vitro. However, replication activity could be restored by mixing the two extracts. Replication activity in either extract also could be restored by addition of appropriate replication-deficient fractions purified from cells infected with wild-type adenovirus. By using such assays, H5ts36- and H5ts125-complementing activities were extensively purified. As expected, purified H5ts125-complementing activity consisted of a single major polypeptide, the 72-kilodalton (kDal) adenovirus DNA binding protein. The purified H5ts36-complementing activity consisted of the 80-kDal adenovirus terminal protein precursor and two other major polypeptides with apparent molecular masses of 140 and 65 kDal. Formation of the 80-kDal terminal protein-dCMP complexes, the proposed initial step in adenovirus DNA replication, required components in the purified H5ts36-complementing fraction and a cellular factor(s) but did not require the adenovirus DNA binding protein. The complete in vitro adenovirus DNA replication reaction was reconstituted from the purified H5ts36-complementing activity, the adenovirus DNA binding protein, and an extract from uninfected cells.

Adenoviruses, Human↗
PubMed1987

Gene expression rather than the initiation of DNA replication is the principal target of lethal u.v.-induced damage in a regulatory region of SV40 DNA.

The survival of transfected simian virus (SV) 40 DNA is acutely sensitive to damage in a 302-bp regulatory region that governs viral gene expression and the initiation of viral DNA replication. We investigated whether the lethal effect of damage in this region is due to the disruption of gene expression or to the inhibition of DNA replication by comparing the survival of damaged viral DNA in CV-1 and cos-1 African green monkey cells. Viral early sequences integrated into the genomic DNA of cos-1 cells complement the growth of virus with defective early genes and were therefore expected to reverse viral sensitivity to lesions that interfere with early gene expression. Our results indicate that viral sensitivity to damage in the regulatory region is almost completely abolished in cos-1 cells. This finding identifies gene expression rather than the initiation of DNA replication as the major target for lethal damage in that portion of the SV40 genome. Sensitivity to damage in the viral late gene region is the same in CV-1 and cos-1 cells, indicating that cos-1 cells are not merely more proficient in host-cell reactivation. Our results allow us to partition the overall lethal effect of DNA damage into sectors, and to assign each sector to the disruption of a particular genetic function.

Animals↗
PubMedDate not supplied

[Effect of weightlessness on the DNA replicative function of rat hepatocytes].

The replicative function of DNA of liver cells of rats exposed to strong stress-effects, e.g. suspension for 2.5 hours a day for 6 days, decreased. The rat studies onboard biosatellites of the Cosmos series have demonstrated that a prolonged exposure to microgravity (up to 22 days) is not a stressogenic factor for the DNA synthetic system of liver cells. The transition from 1 g to microgravity cannot be viewed as a strong stressor either, because the rate of DNA synthesis in liver cells at an early period of adaptation to microgravity remains within the normal limits. However, this parameter decreases significantly during the recovery period following 18-22-day flights. Therefore changes in cellular processes related to the DNA replicating function in hepatocytes should be expected to occur in the postflight period rather than at an early period of adaptation to microgravity.

Animals↗
PubMed1998

[Function of DnaA protein, the initiator for chromosomal DNA replication in E. coli].

DnaA protein is an initiator for chromosomal DNA replication in E. coli. We have examined the function of the protein to answer the following four questions; 1. How DnaA protein is inactivated after DNA replication for the suppression of re-initiation? 2. How DnaA protein is activated for the initiation of DNA replication? 3. Does DnaA protein have functions other than that for DNA replication? 4. Is DnaA protein is a good target for new antibiotics? In this review, I summarize our recent studies for these questions.

Adenosine Diphosphate↗
PubMed1984

Effect of 5-fluoro-2'-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase, replicative DNA polymerase, deoxycytidylate deaminase and CDP reductase activities in human lymphocytes.

The inhibitors of DNA synthesis, 5-fluoro-2'-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0-17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0-28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.

Cells, Cultured↗
PubMed1997

Stringent structural and sequence requirements of the human herpesvirus 6B lytic-phase origin of DNA replication.

The lytic-phase origin of DNA replication from human herpesvirus 6B (HHV-6B oriLyt) contains two binding sites for the origin-binding protein (OBPH6B), both of which are required for DNA replication and which are separated by an AT-rich spacer. We have tested the functional significance of the structural, spatial and sequence characteristics of this spacer element by constructing a series of mutated origin sequences and analysing their replication efficiency. Changes in the sequence composition of length of the spacer resulted in dramatic decreases in replication efficiency. Furthermore, in contrast to what has been observed for herpes simplex virus type 1 (HSV-1) oriS, insertion of a complete helical turn of DNA into the spacer also resulted in abrogation of origin function. These data suggest that the arrangement of OBP sites in HHV-6B oriLyt is stringently constrained in terms of spacing and intervening sequence.

Base Sequence↗