Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Su, Robert T.; DePamphilis, Melvin L.

doi:10.1128/jvi.28.1.53-65.1978

Cited by 81 publications

(52 citation statements)

References 30 publications

(45 reference statements)

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“…UV-crosslinking of proteins to nascent DNA in isolated SV40 chromatin SV40 chromosomes were isolated from nuclei of SV40 infected CV-1 monkey kidney cells 36 h post infection by a method adapted from that of Su and DePamphilis (1978). Four culture plates (240X240 mm) were washed twice with ice-cold isotonic buffer containing 20 mM Tris-HCI, pH 7.5; 137 mM NaCl, 5 mM KCI, 1 mM CaC12, 0.5 mM MgCl2, 250 mM sucrose and I mM phenylmethyl sulfonyl fluoride (PMSF); and once with ice-cold low salt buffer [20 mM HEPES-Na, pH 7.8; 5 mM K-acetate, 0.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), I mM PMSF].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1996

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 1996

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“…These molecules can then separate into circular DNA one genome long containing a short gap in the nascent DNA strand within the termination region (11*) (9,18,33,52). In isolated nuclei or nuclear extracts, termination of DNA replication, which results in the formation of circular, covalently closed, superhelical DNA (I), requires soluble proteins found in the cytosol fraction (16,17,47). In contrast to this view of replicon maturation, other data have been interpreted to support a completely uniform movement of replication forks (3-5, 36, 38), with separation of sibling chromosomes occurring via the formation and subsequent resolution of catenated dimers (48).…”

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Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei

Tapper

Anderson

DePamphilis

1982

J Virol

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The maturation of replicating simian virus 40 (SV40) chromosomes into superhelical viral DNA monomers [SV40(I) DNA] was analyzed in both intact cells and isolated nuclei to investigate further the role of soluble cytosol factors in subcellular systems. Replicating intermediates [SV40(RI) DNA] were purified to avoid contamination by molecules broken at their replication forks, and the distribution of SV40(RI) DNA as a function of its extent of replication was analyzed by gel electrophoresis and electron microscopy. With virus-infected CV-1 cells, SV40(RI) DNA accumulated only when replication was 85 to 95% completed. These molecules [SV40(RI*) DNA] were two to three times more prevalent than an equivalent sample of early replicating DNA, consistent with a rate-limiting step in the separation of sibling chromosomes. Nuclei isolated from infected cells permitted normal maturation of SV40(RI) DNA into SV40(I) DNA when the preparation was supplemented with cytosol. However, in the absence of cytosol, the extent of DNA synthesis was diminished threeto fivefold (regardless of the addition of ribonucleotide triphosphates), with little change in the rate of synthesis during the first minute; also, the joining of Okazaki fragments to long nascent DNA was inhibited, and SV40(I) DNA was not formed. The fraction of short-nascent DNA chains that may have resulted from dUTP incorporation was insignificant in nuclei with or without cytosol. Pulse-chase experiments revealed that joining, but not initiation, of Okazaki fragments required cytosol. Cessation of DNA synthesis in nuclei without cytosol could be explained by an increased probability for cleavage of replication forks. These broken molecules masqueraded during gel electrophoresis of replicating DNA as a peak of 80%o completed SV40(RI) DNA. Failure to convert SV40(RI*) DNA into SV40(I) DNA under these conditions could be explained by the requirement for cytosol to complete the gap-filling step in Okazaki fragment metabolism: circular monomers with

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“…Entire replicative SV40 minichromosomes bearing functionally bound replication proteins can be eluted from nuclei of virus infected cells by hypotonic buffer [19]. The DNA of mammalian chromatin, however, is organized into loops of about 5-150 kb firmly attached to the nuclear matrix [20].…”

Section: Separating a Cell Fraction Containing Dna Bound Proteinsmentioning

confidence: 99%

Analyzing changes of chromatin‐bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells

Betteraey-Nikoleit

Eisele

Stabenow

et al. 2003

European Journal of Biochemistry

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of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G 1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the 'hypoxic preinitiation state', ready to initiate replication as soon as normal pO 2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the 'classical' prereplication complex is already formed during hypoxia.

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Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Cited by 81 publications

References 30 publications

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

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

Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei

Analyzing changes of chromatin‐bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells

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