Oncogene (1997) 14, 1611 ± 1615 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Phosphorylation of DNA polymerase a-primase by Cyclin A-dependent kinases regulates initiation of DNA replication in vitro C Voitenleitner1,2, E Fanning1,2, and H-P Nasheuer1,3 1Institut fuÈr Biochemie, LMU MuÈnchen, WuÈrmtalstr. 221, 81375 MuÈnchen, Germany; 2Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA; 3Institut fuÈr Molekulare Biotechnologie, Abteilung Biochemie, Beutenbergerstrasse 11, 07745 Jena, Germany DNA polymerase a-primase is the only known eukar- of SV40 DNA replication, are potential substrates of yotic enzyme that can start DNA replication de novo.In Cyclin-dependent kinases (Cdks; Dutta and Stillman this study, we investigated the regulation of DNA 1992; McVey et al., 1989; Nasheuer et al., 1991, 1992). replication by phosphorylation of DNA polymerase a- Since RP-A and DNA polymerase a-primase are key primase. The p180 and the p68 subunits of DNA cellular replication proteins, and they are phosphory- polymerase a-primase were phosphorylated using Cyclin lated in a cell cycle-dependent manner, it was suggested A-, B- and E- dependent kinases. This phosphorylation that their phosphorylation might be important for the did not in¯uence its DNA polymerase activity on regulation of DNA replication (Din et al., 1990; Dutta activated DNA, but slightly stimulated primase activity and Stillman 1992; Ferrari et al., 1996; Foiani et al., using poly(dT) single-stranded DNA (ssDNA) without 1995; Nasheuer et al., 1991). changing the product length of primers. In contrast, site- To determine functional interactions of Cdks and speci®c initiation of replication on plasmid DNA replication proteins, we used the in vitro SV40 DNA containing the SV40 origin is aected: Cyclin A-Cdk2 replication system. Four Cyclin-Cdks, i.e. Cyclin A- and Cyclin A-Cdc2 inhibited initiation of SV40 DNA Cdc2, Cyclin A-Cdk2, Cyclin B-Cdc2, and Cyclin E- replication in vitro, Cyclin B-Cdc2 had no eect and Cdk2, phosphorylated DNA polymerase a-primase, but Cyclin E-Cdk2 stimulated the initation reaction. DNA only Cyclin A-Cdc2 and Cyclin A-Cdk2 prevented polymerase a-primase that was pre-phosphorylated by initiation of SV40 DNA replication in vitro.In Cyclin A-Cdk2 was completely unable to initiate the contrast, Cyclin E-Cdk2 and Cyclin B-Cdc2 showed a SV40 DNA replication in vitro; Cyclin B-Cdc2-phos- modest eect. phorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase a-primase remained fully active in the initiation reaction. Results Keywords: DNA replication; cyclins; cyclin-dependent kinases; DNA polymerase a-primase Phosphorylation of DNA polymerase a-primase by Cyclin-Cdk complexes To study the function of protein phosphorylation on Introduction DNA replication, we puri®ed highly active, recombi- nant DNA polymerase a-primase, Cyclin A-Cdc2, Replication of SV40 DNA in vitro serves as an Cyclin A-Cdk2, Cyclin B-Cdc2 and Cyclin E-Cdk2. excellent model system to study eukaryotic DNA The catalytic subunits Cdc2 and Cdk2 phosphorylated replication and its regulation (Challberg and Kelly, neither histone H1 nor DNA polymerase a-primase 1989; D'Urso et al., 1990; Fanning, 1994; Hurwitz et above background level (Figure 1a; data not shown). al., 1990; Li and Kelly, 1984; Stillman, 1989; Waga et The puri®ed Cyclin-Cdk complexes have reproducibly al., 1994). Recently, it was shown that ten proteins or high kinase activity on histone H1 and on DNA protein complexes are necessary and sucient for SV40 polymerase a-primase as a substrate, but they DNA replication in vitro (Hurwitz et al., 1990; Waga et phosphorylated exclusively the p180 and p68 subunits al., 1994) oering the possibility to analyse regulation (Figure 1a; data not shown). Similar activities (adjusted of DNA replication with puri®ed proteins only. with histone H1) of each kinase complex were used to Moreover, biochemical studies of the initiation of phosphorylate DNA polymerase a-primase. Normal- SV40 DNA replication allowed the development of izing kinase activity this way, Cyclin A-Cdk2 kinase an experimental model for the initiation reaction had the highest activity in phosphorylation of the p180 (Challberg and Kelly 1989; Fanning, 1994; Fanning and p68 subunits of DNA polymerase a-primase; and Knippers, 1992; Hurwitz et al., 1990; Stillman, Cyclin A-Cdc2, Cyclin B-Cdc2 and Cyclin E-Cdk2 1989, Wang, 1991). T antigen, RP-A, and DNA complexes were slightly less active in p180 and p68 polymerase a-primase, that are required for initiation phosphorylation (Figure 1a). DNA polymerase a-primase that was pre-incubated with increasing amounts of kinase complexes showed the same DNA polymerase activity on activated DNA Correspondence: H-P Nasheuer as the control enzyme, while its primase activity, that Received 15 February 1996; revised 22 November 1996; accepted 25 November 1996 was measured on poly(dT)-ssDNA, was stimulated up Regulation of DNA replication by phosphorylation C Voitenleitner et al 1612 a Cyclin A-Cdc2 Cyclin A-Cdk2 Cyclin B-Cdc2 Cyclin E-Cdk2 Cdc2 Cdk2 control 195 — — p180 — 130 — 87 — 66 — — p68 — 54 — — p58 — — p48 — 39 — M PP 1 2 3 4 5 6 7 c b ¨ dT18 ¨ dT12 1 2 3 4 5 Figure 1 Phosphorylated DNA polymerase a-primase by Cyclin-Cdk complexes. (a) DNA polymerase a-primase (lane PP) was incubated with puri®ed Cyclin-Cdk complexes, separated by SDS gel electrophoresis, and stained with Coomassie Brilliant-blue. The subunits, that were phosphorylated in the presence of Cyclin A-Cdc2 (lane 1), Cyclin A-Cdk2 (lane 2), Cyclin B-Cdc2 (lane 3), Cyclin E-Cdk2 (lane 4), Cdc2 (lane 5), Cdk2 (lane 6), and control protein (lane 7), were detected by autoradiography. (b) Equal amounts (0.4 units of primase) of DNA polymerase a-primase were pre-incubated with indicated amounts of Cyclin A-Cdc2 (~), Cyclin A-Cdk2 (&), Cyclin B-Cdc2 (*), Cyclin E-Cdk2 (~), Cdk2 (&), or control protein (6), and then primase activities were determined. (c) DNA polymerase a-primase was treated with Cyclin A-Cdk2 or control protein, and then primase products of pre-treated DNA polymerase a-primase on poly (dT) ssDNA were analysed by denaturing gel electrophoresis. Lane 1 and 2 Cyclin A-Cdk2-treated DNA polymerase a-primase (0.2 and 0.4 primase units, respectively), lane 3 and 4 DNA polymerase a-primase treated with control protein (0.2 and 0.4 primase units, respectively), and 0.4 primase units of untreated DNA polymerase a-primase (lane 5) to two- to threefold of that of control enzyme (Figure Cyclin A-Cdc2 and Cyclin A-Cdk2 inhibit initiation of 1b, data not shown). The length of the primase SV40 DNA replication in vitro products was not altered by phosphorylation, since phosphorylated and untreated DNA polymerase a- To study whether the initiation step of replication on primase as well as DNA polymerase a-primase treated SV40-origin containing double-stranded DNA with control protein synthesized equivalent products (dsDNA) is modulated by phosphorylation, Cyclin- (Figure 1c). These data suggest that basic enzymatic Cdk complex or control protein were added directly to functions of DNA polymerase a-primase were not or the initiation reaction. In this assay, Cyclin A-Cdc2 or only moderately altered by phosphorylation with Cdks. Cyclin A-Cdk2 signi®cantly reduced the initiation Regulation of DNA replication by phosphorylation C Voitenleitner et al 1613 2 2 2 2 2 Cyclin A-Cdc Cyclin A-Cdk Cyclin B-Cdc2 Cyclin E-Cdk2 control Cyclin A-Cdk Cyclin B-Cdc Cyclin E-Cdk control active Cdk – + – + – + – + – – – – units 0.4 0.2 0.4 0.2 0.4 0.2 0.4 0.2 0.4 ¨ dT18 ¨ dT18 ¨ dT12 ¨ dT12 1 2 3 4 5 6 7 8 9 10 11 12 Arb units 41 5.3 29 4.9 33 23 25 27 22 22 23 Rel Inc.(%) 18 100 15 100 91100124100 95100 1234 5678910M Figure 2 Initiation of SV40 DNA replication in the presence of Arb units 69 0.7 4 18 41 28 58 30 66 Cyclin-dependent kinases. The initiation reaction of dsDNA in Rel Inc.(%)104 2.3 6 60 63 92 88 100 100 the puri®ed system was performed in the presense of immunoanity puri®ed Cyclin A-Cdc2 (lane 2 and 3), Cyclin Figure 3 Initiation of SV40 DNA replication in vitro by A-Cdk2 (lane 4 and 5), Cyclin B-Cdc2 (lane 6 and 7), Cyclin E- phosphorylated DNA polymerase a-primase. For comparing the Cdk2 (lane 8 and 9), or proteins from mock infected cells that initiation activity of the DNA polymerase a-primase complexes, bind to 12CA5 antibodies (control protein, lane 10 and 11). In primase activity of each DNA polymerase a-primase was parallel, the initiation reactions were performed with either active measured shortly before the initiation assay. The initiation (lanes 2, 4, 6 and 8), heat inactivated (10 min at 708C) kinase products of 0.4 units of untreated immunoanity-puri®ed DNA complexes (lanes 3, 5, 7 and 9), or with control protein polymerase a-primase were analysed (lane 1). In parallel, 0.2 and (untreated, lane 10, and heat treated, lane 11). Reaction products 0.4 primase units of Cyclin A-Cdk2- (lane 2 and 3), Cyclin B- were analysed by denaturing gel electrophoresis and autoradio- Cdc2- (lane 4 and 5), or Cyclin E-Cdk2- (lane 6 and 7) graphy. The initiation products of 0.4 units of untreated DNA phosphorylated DNA polymerase a-primase, or DNA polymer- polymerase a-primase are shown in lane 1. Radioactive material ase a-primase treated with control protein (lane 8 and 9) were that is detectable in the absence of DNA polymerase a-primase is incubated under identical conditions and the initiation products were analysed.