Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus

Nakamura, Hiromu; Morita, Toshiteru; Sato, Chikara

doi:10.1016/0014-4827(86)90583-5

Cited by 412 publications

(290 citation statements)

References 18 publications

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“…2). This is in line with a model for the dynamic structural organization of replicon domains in the mammalian nucleus (25,27), in which multiple replication origins are assembled together on the nucleoskeleton, just before initiation of replication, and DNA loops of replicon size are arrayed around them. Starting from the center, these structures are then gradually "filled" with newly synthesized DNA.…”

Section: Discussionsupporting

confidence: 85%

“…Since nuclear area correlates with DNA content (9,371, nuclear areas of labeled cells were measured in five cell lines. We also determined the frequencies of the five staining patterns in pulse-labeled MCF7 cells at several time intervals after recruitment of cell populations from a Tamoxifen-induced Gl,o-block (25) and compared these frequencies with flow cytometric DNA histograms. As shown in Figure 4, the BrdUrd-labeling index increased from 14.5% to 70% during the first 17 h after replenishement of the culture medium.…”

Section: Resultsmentioning

confidence: 99%

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Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

et al. 1989

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After pulse labeling of mammalian cells in vivo or in vitro with 5-bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA-incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidasestained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms.The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kine tics.Key terms: DNA replication, B-bromodeoxyuridine, monoclonal anti-BrdUrd antibodies, interphase nucleus There is much evidence from (3H)-thymidine (3HTdR) labeling studies that DNA synthesis in an eukaryotic nucleus consists of an ordered cascade of initiations of replicons (4), controlled by an intranuclear mechanism (36). This is particularly clear from studies on polytene chromosomes of dipteran larvae, describing various patterns of DNA synthesis, tentatively assigned to specific periods of the S-phase (1,2,28). In general, GC-rich euchromatin replicates early, whereas AT-rich heterochromatic DNA replicates late (13); these early and late DNA synthesizing sites roughly correspond to R-and G-bands on trypsinGiemsa-stained chromosomes, respectively (6). However, also in euchromatin, distinct early-and late-replicating DNA fractions have been decribed (24); Goldman et al. (1 1) have presented evidence that genes that are obligatory or potentially active in a given cell type replicate early in that cell type, whereas genes that are permanently inactive replicate late.The characteristic grain distributions observed in eukaryotic nuclei after in situ autoradiography of incorporated 3HTdR (35) may reflect both the structural organization of replicon domains and a nonrandom localization of chromosomes in the interphase nucleus (12,161. In particular, centromeric and other highly repeated nontranscribed sequence domains may be positioned on the nucleolus or nuclear membrane and act as general organizing centers for cell type-specific interphase patterns (21). Multiple adjacent replicons appear to be clustered on fixed sites of the nuclear matrix (17,271, and it has been suggested that clustered replication origins are activated simultaneously (25). Early DNA synthesis then seems to occur in replicon clusters evenly distributed over the nucleus, whereas late DNA synthesis, being primarily heterochromatic, is preferentially restricted to domains on the periphery of the nucleus and in nucleoli (35,361. However, the assignment of autoradiographic labeling patterns to spe...

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Section: Discussionsupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

et al. 1989

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“…The intranuclear location of PCNA overlaps with that of DNA polymerase a and BrdUrd in normally growing cells treated with detergent (3,7,21). This fact supports the idea that PCNA is part of a complex corresponding to the replisome (9,22,30).…”

Section: Discussionsupporting

confidence: 74%

“…Assuming that PCNA is bound exclusively to the initiation sites of DNA replication in cells accumulated a t GUS boundary by HU, another question arises: Did the elevation of PCNA content by HU treatment result from the increase in the number of sites bound with PCNA or in the amount of PCNA per site? It is well known that the number of DNA replication sites is much less in cells just leaving G1 phase than in those in m i d 3 phase (21). This has been supported by the flow cytometric analysis of BrdUrd labeled cells.…”

Section: Discussionmentioning

confidence: 81%

Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it

1993

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Flow cytometric bivariate DNA/PCNA analysis was performed to investigate the expression of PCNA during the cell cycle and the implication in DNA replication in HeLa cells, using a monoclonal antibody (PC10) to PCNA. The expression of PCNA was evident in almost all cells growing exponentially, when cells were fixed in methanol. The total amount of PCNA altered a little during the cell cycle. However, the treatment with Triton X‐100 extracted 80–89% of total PCNA from the cells, resulting in the dramatic change of bivariate DNA/PCNA distribution pattern. PCNA was completely removed from nuclei in both G1 and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S phase nuclei. After the treatment of cells with Triton X‐100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribution pattern in cells treated with Triton X‐100 was strikingly so similar to the DNA/BrdUrd distribution pattern that it was unable to differentiate one from the other. It is concluded that the detergent treatment of cells allows the rapid analysis of the cell cycle.The inhibition of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA content mainly due to the increase in the fraction of the detergent extractable PCNA. It was apparent, however, that in cells incubated with Triton X‐100, the pattern of the bivariate DNA/PCNA distribution was not basically different from that in cells without HU treatment. The level of PCNA bound to nuclear structures (PCNA not extracted with detergent) increased in cells arrested at the G1/S boundary with the time of hydroxyurea treatment. Eventually, it became difficult to differentiate between cells in the G1/S boundary and the early S phase on the basis of DNA and PCNA contents. However, the increase in bound PCNA was minimal in S phase cells. The observation suggests that PCNA is bound to the potential initiation sites of DNA replication together with DNA replication associated enzymes in advance of the commencement of DNA replication and that it is never associated with late replicating DNA without the duplication of early replicating DNA. © 1993 Wiley‐Liss, Inc.

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“…Some of these cells had a positive p53 stain only in their cytoplasm (indicated by the thin arrow-head in Figure 7a). A higher magni®cation revealed that the nuclear p53 protein appeared in clusters with an intense signal near the nuclear envelope (Figure 7b), suggesting the possibility that the p53 protein might co-localize with the DNA replication apparatus, which are tightly clustered in foci anchored to the nuclear membrane (Nakamura et al, 1986;Mills et al, 1989;Cox and Laskey, 1991;Hozak et al, 1993;Coverley and Laskey, 1994). These results are in agreement with the observations by others that the p53 protein may be associated with DNA replication activity (Gannon and Lane, 1987;Cox et al, 1995).…”

Section: Association Of the Wt P53 Protein With Dna Replication Activmentioning

confidence: 98%

Excision of mismatched nucleotides from DNA: a potential mechanism for enhancing DNA replication fidelity by the wild-type p53 protein

Huang

1998

Oncogene

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The tumor suppressor p53 plays a critical role in the regulation of the cell cycle and the maintenance of genetic stability. The 3'?5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule, but the biological signi®cance of this activity remains elusive. Using an in vitro DNA replication assay with puri®ed human DNA polymerases, p53 protein, and de®ned DNA primer/templates, we demonstrated that the wild-type (wt) p53 protein, but not the mutant p53 protein, speci®cally enhanced the DNA replication ®delity of polymerase (pol) a, an enzyme that lacks 3'?5' exonuclease activity. The misincorporation of non-complementary deoxynucleotides into DNA by pol a was substantially decreased by p53. In contrast, wt p53 showed no signi®cant e ect on replication ®delity or the rates of DNA synthesis by human pol e or the bacterial enzyme pol I. Inhibition of 3'?5' exonuclease activity by guanosine monophosphate (GMP) abolished the ability of p53 to enhance the replication ®delity of pol a. Quantitative analyses revealed that the 3'?5' exonuclease activity of p53 e ectively removed mismatched nucleotides from DNA in preference over matched nucleotides. Furthermore, study in intact cells revealed that the wt p53 protein was co-localized with DNA synthesis activity in S phase cells. These results suggest a possibility that the 3'?5' exonuclease of the wt p53 protein might provide the proof-reading function for DNA pol a. The preferential excision of mismatched nucleotides from the replicating DNA strand appears to be a potential biochemical mechanism by which p53 enhances DNA replication ®delity and thereby helps to maintain genomic integrity.

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Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus

Cited by 412 publications

References 18 publications

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it

Excision of mismatched nucleotides from DNA: a potential mechanism for enhancing DNA replication fidelity by the wild-type p53 protein

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