Nuclear DNA polymerases and the HeLa cell cycle.
Purified nuclei of HeLa S3 cells contain two DNA-dependent DNA polymerases that have distinct physical and enzymatic properties. We have investigated the variations in their activity during the cell cycle of a synchronized culture. Cells were synchronized by a double thymidine block, harvested at various phases of the cycle, and the two DNA polymerases were purified partially by DEAE-cellulose and phosphocellulose chromatography. The activity of DNA polymerase I (low molecular weight, N-ethylmaleimide-insensitive) remains essentially constant throughout the cycle. The activity of DNA polymerase II (high molecular weight, N-ethylmaleimide-sensitive), however, increases during G1 to mid-S and declines, 7- to 10-fold between late-S and G2. Addition of cycloheximide (60 mug/ml) to cultures 12 hours after the release from thymidine block abolishes the rise in the activity of DNA polymerase II. Cycloheximide also reduced the activity of DNA polymerase I by 60%. Addition of hydroxyurea (1mM) at 1 hour after release has no effect on the activity of either enzyme. We conclude that in HeLa cells, DNA polymerase I and II are distinct enzymes, that DNA polymerase II probably functions in DNA replication and is probably induced in response to stimuli for DNA biosynthesis.
D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D003513:Cycloheximide / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006367:HeLa Cells / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006918:Hydroxyurea / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D013698:Templates, Genetic
D007527:Isoenzymes; D003513:Cycloheximide; D004254:DNA Nucleotidyltransferases; D008274:Magnesium; D005033:Ethylmaleimide; D006918:Hydroxyurea