Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states

Abstract

DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. alpha-Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE-cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, alpha- and beta-polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late-passage and postconfluent early passage fibroblasts, amounts of alpha-polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The alpha-polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCI and could be inhibited by a monoclonal IgG fraction prepared against KB cell alpha-polymerase. By several criteria, there was no significant differences in levels of UV-stimulated repair synthesis observed in early or late-passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of alpha-polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for alpha-polymerase from late-passage cells may be attributed more generally to the nondividing nature of these cells.

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

“…This increase in activity supports the suppositions that DNA polymerase a is the major DNA replication enzyme in mammalian cells and that its activity is coordinately regulated with the initiation of DNA synthesis. In contrast, other studies demonstrate that the majority of DNA polymerase a activity does not change with DNA synthesis or repair (Foster & Collins, 1985;Krauss & Linn, 1986). Potential causes of this discrepancy are the methods of extract preparation and, most importantly, the means by which cells are synchronized.…”

Radicals in Berkeley?

“…After my return to Berkeley, I continued to study this phenomenon and finally concluded that what we were seeing was the replacement of the accurate, replicative polymerases with the less faithful, DNA repair "sloppier copier" lesion bypass enzymes (7). However, in the process, we also discovered a new replication DNA polymerase, polymerase ⑀, which we went on to characterize (8).…”