Cell cycle dependent activities of DNA polymerases .alpha. and .delta. in Chinese hamster ovary cells

Abstract

The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.

Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle.

“…DNA polymerase 8 has not yet been enzymologically, physicochemically, or genetically defined, and its relation to polymerase a is not clear; however, antibodies specific for each of the two enzymes do not cross-react (11,24,48). One recent report describing the enzymatic activities of both DNA polymerases a and 8 in cell lysates from phase-specific populations of cycling cells failed to show significant variation in enzymatic activities of either polymerase (27). In this study we investigated the molecular mechanisms that are involved in the expression of DNA polymerase a in cell transformation, in activation of cell proliferation from quiescence, and during the cell cycle.…”

Reduction of DNA primase activity in aging but still proliferating cells

“…Aphidi-Colin, a potent inhibitor of DNA polymerases a and 8, inhibited whole cell lysates by 96% and nucleoids by 98%, indicating that most of the activity being measured is that of DNA polymerase (Y or 6 . The monoclonal antibody designated SJK 132-20, which inhibits DNA polymerase alpha but not DNA polymerase delta (Marraccino et al, 1987), inhibited whole cell lysates by 88%, and nucleoids by 91%, indicating only a slight contribution by polymerase 8. Thus, the conditions appear adequate for estimations of DNA polymerase (Y activity .…”

“…The monoclonal antibody designated SJK 132-20, a DNA polymerase a neutralizing antibody (Tanaka et al, 1982) was purified from its hybridoma cell line exactly as previously described (Collins et al, 1989). cells (Marraccino et al, 1987) and HeLa cells (Collins and Chu, 1987). However, total DNA primase activity appears to be elevated during S-phase (Philippe et al, 1986;Vishwanatha et al, 1986;Collins and Chu, 1987).…”

Contribution of DNA polymerase δ to DNA replication in permeable CHO cells synchronized in S phase

“…However, the recent discovery of DNA polymerase S has raised an important but unsettled question about the relationship of these two polymerases and their roles in DNA replication (2). Recent studies have indicated that both polymerases may be involved in the replication of mammalian chromosomes (3)(4)(5)(6). but there has been some dispute over their relative contributions.…”