Anti-human DNA polymerase a murine IgG SJK-287-38 [Tanaka, S., Hu, S.-Z., Wang, T. S. These data suggest that fluctuation in DNA polymerase a activity is not due to changes in DNA polymerase a protein content, or nuclear translocation, but that another mode of enzyme regulation, such as posttranslational modification, might be involved.Detailed investigations of posttranslational alterations of DNA polymerase a have not been carried out for several reasons. DNA polymerase a shows heterogeneity with respect to both size (4, 5) and oligoenzyme association (6). Furthermore, DNA polymerase a exists in low abundance, with estimates of 20,000-60,000 molecules per cell (7). Recent development of immunological probes to DNA polymerase a provides a means to ask whether posttranslational phosphorylation/dephosphorylation modifications of this enzyme occur in vivo. In this study, we asked whether DNA polymerase a was phosphorylated in exponentially growing rat embryonic cells expressing pp60src, the tyrosine-specific protein kinase and transforming function of Rous sarcoma virus and, if so, whether the enzyme activity was altered by phosphorylation/dephosphorylation reactions.
MATERIALS AND METHODSCell Culture. Rat-l(tsLA24/RSV) cells (rat embryonic fibroblasts infected with a Rous sarcoma virus mutant that is temperature-sensitive for the transformed phenotype) were grown in monolayer culture at the permissive temperature (35°C) as described (8). Hybridoma cells producing monoclonal antibody to DNA polymerase a (SJK-287-38) were obtained from the American Type Culture Collection and grown as directed.For labeling exponentially growing cultures with [32P]orthophosphate, cells were seeded at 6 x 106 cells per 150-mm dish (Falcon). Forty hours later, the cells were labeled for 6 hr at 350C with [32P]orthophosphate (HCl-free, ICN) at 1.0 mCi/ml (1 Ci = 37 GBq) in 5.0 ml of phosphate-free McCoy's SA medium plus 10% fetal bovine serum per plate. Cellular proteins were radiolabeled by incubating cultures for 6 hr in 5 ml of methionine-free McCoy's SA medium containing 10% fetal bovine serum and 100 ,tCi of [35S]methionine (1000 Ci/mmol, New England Nuclear) per ml.DNA Polymerase a Activity Assay. The incorporation of [methyl-3H]dTTP (77.0 Ci/mmol, New England Nuclear) into bovine pancreatic DNase I-activated salmon sperm DNA (Worthington) was measured as described (9). One unit of enzyme activity is defined as 1 nmol of [3H]dTMP incorporated into product per hour at 37°C.Neutralization and Immunoprecipitation of Rat-1(tsLA24/ RSV) DNA Polymerase a by Anti-Human Polymerase a IgG SJK-287-38. Highly purified monoclonal antibody to human DNA polymerase a was prepared as described (10,11).Cells were swelled in 50 mM Tris-HCl, pH 7.5/1 mM EDTA/5 mM MgCI2/1 mM 2-mercaptoethanol/1 mM phenylmethylsulfonyl fluoride with 10% (vol/vol) glycerol, or without glycerol, for 30 min on ice, disrupted by sonication, and then centrifuged at 31,000 x g for 30 min at 40C. After centrifugation in sucrose gradients, fractions were adjusted to 0...