Binding of the DNA polymerase .alpha.-DNA primase complex to the nuclear matrix in HeLa cells

Collins, James M.; Chu, Annie K.

doi:10.1021/bi00392a004

Cited by 31 publications

(19 citation statements)

References 44 publications

(79 reference statements)

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“…As shown in Table V, soluble DNA pol ␣ activity remained stable up to 6 h after serum dilution and changed relatively little, being doubled after 30 h, in agreement with previous data [Foster and Collins, 1985;Fry and Loeb, 1986;Vishwanatra et al, 1986;Collins and Chu, 1987;Wang, 1991], and conceivably representing the effect of de novo synthesis of the enzyme [Miyazawa et al, 1993]. Nuclear DNA pol ␣ activity started to rise at 6 h after dilution and then gradually increased, reaching a plateau value at 22 h. Overall, this activity showed a fourfold increase at 30 h. A striking increase in nuclear matrix-bound DNA pol ␣ activity was seen as early as 2 h after dilution.…”

Section: Dna Pol ␣ and Dna Primase Activities In Subcellular Fractionsupporting

confidence: 91%

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

Martelli

Capitani²,

Neri³

1998

J. Cell. Biochem.

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We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G1 phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18-22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [3H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activities was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures.

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Section: Dna Pol ␣ and Dna Primase Activities In Subcellular Fractionsupporting

confidence: 91%

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

Martelli

Capitani²,

Neri³

1998

J. Cell. Biochem.

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“…Numerous studies have suggested that there must be an organizing structure such as the nuclear matrix that helps coordinate spatial regulation of DNA synthesis (Dijkwel et al 1979;Berezney and Buchholtz 1981;Hunt and Vogelstein 1981;Carri et al 1986;Collins and Chu 1987;Vaughn et al 1990;Neri et al 1992;Hozak et al 1993). Components of the nuclear substructure that have been implicated in establishing the pattern of DNA replication sites are the nuclear lamins.…”

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confidence: 99%

Nuclear organization of DNA replication in primary mammalian cells

Kennedy¹,

Barbie²,

Classon³

et al. 2000

Genes Dev.

270

240

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Using methods that conserve nuclear architecture, we have reanalyzed the spatial organization of the initiation of mammalian DNA synthesis. Contrary to the commonly held view that replication begins at hundreds of dispersed nuclear sites, primary fibroblasts initiate synthesis in a limited number of foci that contain replication proteins, surround the nucleolus, and overlap with previously identified internal lamin A/C structures. These foci are established in early G 1 -phase and also contain members of the retinoblastoma protein family. Later, in S-phase, DNA replication sites distribute to regions located throughout the nucleus. As this progression occurs, association with the lamin structure and pRB family members is lost. A similar temporal progression is found in all the primary cells we have examined but not in most established cell lines, indicating that the immortalization process modifies spatial control of DNA replication. These findings indicate that in normal mammalian cells, the onset of DNA synthesis is coordinately regulated at a small number of previously unrecognized perinucleolar sites that are selected in early G 1 -phase.

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“…Hence, the amount of bound pol a was essentially constant through S phase, suggesting a fairly constant rate of DNA replication through S phase. This finding is not in agreement with a local minimum of bound pol a in mid-S phase (8,12) and not consistent with a local minimum in the DNA replication rate in mid-S phase (7,12). Assuming that the pol a molecules replicate the lagging strand and an equal number of pol 8 molecules replicate the leading strand (10,32,33), we calculated a chain elongation (9) 0.94 ± 0.06 1.07 ± 0.07 0.99 ± 0.08 a Relative to the average for S-phase nuclei.…”

mentioning

confidence: 87%

“…On the other hand, after paraformaldehyde fixation, pol a is localized to the nucleus of all interphase cells in exponentially growing cultures (1,18). Other studies indicate that a fraction of pol a is bound to the nuclear matrix (8,9,12,27,28,36,44); the association seems to be S-phase specific (8,12,27). In this study, we have used flow cytometry and confocal laser scanning microscopy to determine the expression, nuclear binding, and localization of pol a in resting and proliferating cells.…”

mentioning

confidence: 99%