Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration.

The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.

Section: Resultssupporting

confidence: 93%

Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells

Reddy

Reed²,

Sheehan³

et al. 1992

“…Since replication is proposed to occur at the level of the nuclear matrix (Smith & Berezney, 1982;Pardoll et al, 1980;Valenzuela et al, 1983;Foster & Collins, 1985), the preponderance of the DNA polymerase -DNA primase complex at that level, as a reflection of greater demands for chain initiation, might be expected. In this regard, while this paper was under review, Tubo and Berezney (1987) reported that rat liver nuclear matrix retains ribonuclease H activity, 3' to 5' exonuclease activity, and DNA methylase activity in addition to DNA polymerase a and DNA primase. They observed an increase in the amount of DNA polymerase a, ribonuclease H, DNA methylase, and DNA primase bound to the nuclear matrix before entry into the DNA synthetic phase, and thus, our data confirm theirs, with respect to DNA primase, in a different system.…”

Section: Resultsmentioning

confidence: 97%

“…DNA primase activity was found to correlate with DNA synthesis in spleen and cardiac muscle cells during postnatal development . The activity of the DNA polymerase a-DNA primase complex increased during liver regeneration (Philippe et al, 1986;Tubo & Berezney, 1987). The amount of the DNA polymerase -DNA primase complex purified from synchronized cells was 8-fold greater during the S phase of the cell cycle than during G2 (Vishwanatha et al, 1986).…”

mentioning

confidence: 97%

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

Collins¹,

Chu²

1987

It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

“…Intracellular Ca2+ and CaM, increased in the late G1 period prior to the onset of DNA synthesis (S phase), have been shown to translocate to the nucleus and associate with the nuclear matrix (Pujol et al, 1989;Serratosa et al, 1988). Nuclear matrix is suggested to be the site for newly replicating DNA and contains the enzymes of DNA synthesis assembled into complex (Nelson et al, 1986;Tubo & Berezney, 1987). More recently, it was demonstrated that CaM-specific monoclonal antibodies markedly inhibit DNA replication in permeabilized fibroblasts (Reddy et al, 1992b).…”

Section: Discussionmentioning

confidence: 99%

Growth factor modulated calmodulin-binding protein stimulates nuclear DNA synthesis in hemopoietic progenitor cells

Gp¹,

Reed²,

Dh³

et al. 1994

A specific calmodulin-binding protein of 68 kDa (CaM-BP68) is modulated in response to growth factors that induce proliferative stimulation in a variety of hemopoietic progenitor cells. The nuclear localization of the CaM-BP68 coincided temporally with interleukin 3 (IL-3)-dependent progression of synchronized FDC-P1 cells from G1 to S phase [Reddy et al. (1992) Blood 79, 1946-1956]. To delineate the role of the CaM-BP68 in the onset of DNA synthesis (S phase), this protein was purified to an apparent homogeneity from FDC-P1 cells and its effects on DNA replication in permeabilized FDC-P1 cells were examined. Purified CaM-BP exhibited a single silver-stained protein band of 68 kDa on SDS-polyacrylamide gels. This purified protein, when incubated with permeabilized log-growing FDC-P1 cells, caused a 3-4-fold increase in the rate of [3H]dTTP incorporation into DNA as compared to the controls. There was a direct correlation between the increase in the rate of [3H]dTTP incorporation into DNA and the concentration of the added CaM-BP68 in the incubation mixture. These observations suggest that the CaM-BP68, whose nuclear localization is associated with growth factor dependent proliferative stimulation of myeloid progenitor cells, is involved in the regulation of nuclear DNA synthesis.