Origin-Specific Initiation of Mammalian Nuclear DNA Replication in aXenopusCell-Free System

Wu, Jianpeng; Yu, Guanhua; Gilbert, David M.

doi:10.1006/meth.1997.0530

Cited by 43 publications

(97 citation statements)

References 39 publications

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“…To synchronise cells in S-phase, cells were first synchronised in mitosis as described previously (Gilbert and Cohen, 1987). Selected mitotic cells were plated onto coverslips in fresh medium, and 3 hours later 400 µM mimosine (Sigma) was added to the medium for an additional 10 hours to block cells at the G1/S border (Wu et al, 1997). Cells were then released into S-phase by replacing with warm medium, as previously described (Wu et al, 1997).…”

Section: Antibody Production and Characterisationmentioning

confidence: 99%

“…Selected mitotic cells were plated onto coverslips in fresh medium, and 3 hours later 400 µM mimosine (Sigma) was added to the medium for an additional 10 hours to block cells at the G1/S border (Wu et al, 1997). Cells were then released into S-phase by replacing with warm medium, as previously described (Wu et al, 1997). At various times after release into S-phase, cells were pulselabelled with 30 µg/ml 5′-bromo-2′-deoxyuridine (BrdU) for 10 minutes and then fixed in 2% paraformaldehyde for 10 minutes at room temperature.…”

Section: Antibody Production and Characterisationmentioning

confidence: 99%

“…Anti-MeK20H4 antibody was diluted 1:100 and detected by an Alexa 488-conjugated Goat anti-rabbit IgG (Molecular Probes; diluted at 1:200). The bound antibodies were next fixed with 4% formaldehyde in PBS for 15 minutes to preserve them during denaturation and then washed in 0.5% NP40 in PBS for 15 minutes before detection of BrdU according to the method of Wu et al (Wu et al, 1997). DNA was stained with 10 ng/ml DAPI and cover slips were mounted in Vectashield (Vector Laboratories).…”

Section: Antibody Production and Characterisationmentioning

confidence: 99%

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Heterochromatin andtri-methylated lysine 20 of histone H4 in animals

Kourmouli

Jeppesen

Mahadevhaiah³

et al. 2004

Journal of Cell Science

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225

181

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Tri-methylated lysine 20 on histone H4 (Me(3)K20H4) is a marker of constitutive heterochromatin in murine interphase and metaphase cells. Heterochromatin marked by Me(3)K20H4 replicates late during S phase of the cell cycle. Serum starvation increases the number of cells that exhibit high levels of Me(3)K20H4 at constitutive heterochromatin. Me(3)K20H4 is also present at the centromeric heterochromatin of most meiotic chromosomes during spermatogenesis and at the pseudoautosomal region, as well as at some telomeres. It is not present on the XY-body. During murine embryogenesis the maternal pronucleus contains Me(3)K20H4; Me(3)K20H4 is absent from the paternal pronucleus. On Drosophila polytene chromosomes Me(3)K20H4 is present in a 'punctate pattern' at many chromosomal bands, including the chromocenter. In coccids it is present on the facultatively heterochromatinised paternal chromosome set. We also present evidence that Me(3)K20H4 is dependent upon H3-specific Suv(3)9 histone methyltransferase activity, suggesting that there may be 'epigenetic cross-talk' between histones H3 and H4

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Section: Antibody Production and Characterisationmentioning

confidence: 99%

Section: Antibody Production and Characterisationmentioning

confidence: 99%

Section: Antibody Production and Characterisationmentioning

confidence: 99%

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Heterochromatin andtri-methylated lysine 20 of histone H4 in animals

Kourmouli

Jeppesen

Mahadevhaiah³

et al. 2004

Journal of Cell Science

Self Cite

225

181

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“…The discrepancy between the in vivo and the in vitro replication results with total extracts may be related to the absence of nuclear structure in vitro. An intact nuclear structure has been suggested to be required for the initiation of DNA replication in human (Krude et al, 1997), yeast (Pasero et al, 1997) and Xenopus (Wu et al, 1997;Fig. 7.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the DNA replication competence of thexrs-5 mutant cells defective in Ku86

Matheos

Novac

Price

et al. 2003

Journal of Cell Science

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The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein,previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2,PCNA, DNA polymerase ϵ and δ, Primase and Topoisomerase IIα)were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oriβ) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oriβ origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells.

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“…Mitotic cells were obtained by mechanical shake-off after 4-5 h incubation with nocodozole (Sigma Aldrich) at 50 ng/ml as described [21]. G 1 /S-phase cells were prepared by releasing mitotic cells in the medium for 4-5 h and then adding 1 mM hydroxyurea (HU) for another 10-11 h.…”

Section: Cell Culture and Synchronymentioning

confidence: 99%

Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

Ren

2004

Cell Res

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DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early-or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chk1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-Sphase checkpoint blocks the DNA replication progression in the late stage of S phase.

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Origin-Specific Initiation of Mammalian Nuclear DNA Replication in aXenopusCell-Free System

Cited by 43 publications

References 39 publications

Heterochromatin andtri-methylated lysine 20 of histone H4 in animals

Heterochromatin andtri-methylated lysine 20 of histone H4 in animals

Analysis of the DNA replication competence of thexrs-5 mutant cells defective in Ku86

Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

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