Biochem. J. (1998) 335, 581–588 (Printed in Great Britain) 581 Mutant DNA polymerase δ from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen ' > Mylene PERDERISET*, Giovanni MAGA†, Karine PIARD*, Stefania FRANCESCONI*, Isabelle TRATNER*, Ulrich HUBSCHER† and Giuseppe BALDACCI*1 *CNRS-IFC1, Institut de Recherche sur le Cancer, UPR 9044, 7 Rue Guy Moquet BP8, 94801 Villejuif, France, and †Institut fu$ r Veterina$ rbiochemie, Universita$ tZu$rich- Irchel, Winterthurerstrasse 190, CH-8057 Zu$ rich, Switzerland We have isolated and characterized DNA polymerase δ (pol δ) In contrast, Vmax values are lower than the wild-type value. The from two thermosensitive Schizosaccharomyces pombe strains, major in itro defect appears to be decreased stimulation of polδts1 and polδts3, mutated in two different evolutionarily mutant enzymes by PCNA, resulting in reduced velocity of DNA conserved domains of the catalytic subunit. At the restrictive synthesis. In addition, ts1 pol δ is not stimulated by low PCNA temperature of 37 mC polδts1 and polδts3 mutant strains arrest concentration at 37 mC, although low concentrations stimulate growth in the S phase of the cell cycle. We show that at low levels activity at 25 mC, suggesting that this thermolability at low levels of primer ends, in itro stimulation by proliferating cell nuclear of primer ends could be its critical defect in io. Thus, both ts1 antigen (PCNA) of mutant enzymes is lower than stimulation of and ts3 pol δ mutations are located in regions of the catalytic wild-type pol δ. Affinity for primer (3«-OH) ends and processivity subunit that seem necessary, directly or indirectly, for its efficient of mutant enzymes do not appear different from wild-type pol δ. interaction with PCNA. INTRODUCTION [10,14]. Pol δ holoenzyme from one of the S. cereisiae mutant strains showed thermolabile activity in itro, but the precise DNA polymerase (pol) δ is a eukaryotic enzyme that is essential location of the mutation in p125 is unknown [15]. At restrictive for in itro replication of DNA containing the origin of replication temperatures S. pombe pol δ mutant strains arrest their growth in of the simian virus 40 (SV40) [1]. Pol δ holoenzyme from the S phase of the cell cycle with a DNA content comprised of Schizosaccharomyces pombe is believed to consist of five subunits between 1C (where 1C is the DNA content of haploid nuclei) and (125, 55, 54, 42 and 22 kDa). The catalytic subunit (p125) is 2C [14,16]. Two of the strains, polδts1 and polδts3, are mutated encoded by the pol3+ gene [2], p55 and p54 are encoded by cdc1+ in two different evolutionarily conserved domains of p125 [14,16]. and cdc27+ genes respectively [3], and p42 and p22 (encoded by + The polδts1 allele has two adjacent point mutations in the NT1 the cdm1 gene) have been isolated recently and their function is "%$ "%% domain: Ala ! Val and Pro ! Ser. The polδts3 allele con- still unclear [4]. Genetic and biochemical studies have shown that + tains a single amino acid change in the ZnF2 domain (a putative p125 interacts with the product of the cdc1 gene, and that the "!'% Zn finger) [17] at the C-terminus of p125: Arg ! Gln. We products of cdc27+ and cdc1+ genes interact together, suggesting characterized mutant p125 from these strains in order to obtain that p125, p55 and p54 form a complex [3]. Recombinant p125 insights into the function of domains NT1 and ZnF2. In this expressed in insect cells or in Escherichia coli is not activated by paper, we report the partial purification and comparative analysis proliferating cell nuclear antigen (PCNA) [5,6]. Indeed, the p48 of pol δ from wild-type and from mutant S. pombe strains polδts1 subunit of human pol δ (homologous to S. pombe p55) is and polδts3. We show that at low levels of primer ends, essential for stimulation of polymerase activity by PCNA, stimulation of both mutant enzymes by PCNA is lower than for indicating that functional p125–PCNA interaction requires ad- wild-type pol δ, and that ts1 pol δ displays a temperature- ditional proteins [7–9]. dependent reduction of stimulation by PCNA. In budding and fission yeast the genes encoding the catalytic subunit of pol δ are essential for cell growth [10]. In addition, mutations affecting the 3« ! 5« exonuclease activity of Sac- charomyces cereisiae pol δ result in an increased mutation rate EXPERIMENTAL in io [11]. It has also been proposed that the N-terminal region Materials of S. cereisiae p125 is necessary for a productive association with PCNA [12,13]. Accordingly, a mutant in the NT1 domain Terminal deoxynucleotidyltransferase, E. coli pol I Klenow of human p125 did not show stimulation by PCNA [13]. fragment, nucleotides and ribonucleotides were obtained from Mutations in the gene encoding p125 that induce thermosensitive Boehringer Mannheim. Phosphocellulose (P11), glassmicrofibre growth have been isolated in both budding and fission yeast filters (GF}C) and DEAE-81 cellulose filters were purchased from Abbreviations used: pol, DNA polymerase; DTT, dithiothreitol; TCA, trichloroacetic acid; SV40, simian virus 40; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen. 1 To whom correspondence should be addressed (e-mail baldacci!infobiogen.fr). 582 M. Perderiset and others Whatman BioSystems Inc. The TSK DEAE 5 PW HPLC column a (7.5¬75 mm) TSK DEAE 5 PW HPLC column equilibrated $ −" was from Beckman Instruments. [ H]dATP (17.5 Ci mmol ) with buffer B containing 25 mM KH#PO%, pH 7.0}10% (v}v) $ was from Du Pont-New England Nuclear, [ H]dTTP glycerol}2 mM EDTA}1 mM DTT}2 mM benzamidine}1 " $ " $# (58 Ci mmol− ) was from ICN, [ H]dCTP (25 Ci mmol− ), [ P]- µg}ml pepstatin A}1 µg}ml leupeptin}5 kallikrein units}ml " dATP (3000 Ci mmol− ) and ECL (enhanced chemiluminescence) aprotinin. Proteins were eluted with three linear KCl gradients Western blotting detection reagents were from Amersham Life (28 ml of 0–200 mM KCl, 12 ml of 200–350 mM KCl and 8 ml of Sciences, poly(dA)400 and oligo(dT)12–18 from Pharmacia 350–500 mM KCl). For quantification analyses, a preparative Biotech, Nitrocellulose (Biotrace NT) was from Gelman Sciences TSK DEAE 5PW column (21.5¬150 mm) was used as the first S. A., and opti-fluor O scintillation cocktail was from the chromatographic step, as described [20], and eluted pol δ was Packard Instrument Company. loaded on a phosphocellulose column as described above. Nucleic acid substrates pol assays Poly(dA) was mixed with oligo(dT) at a molar ratio of 1:3, Replication factor C (RF-C)-independent pol assay as described previously [18]. Single-stranded M13(mp11) DNA was prepared as described previously [19]. For the exonuclease pol activity was assayed with poly(dA)}oligo(dT) as template} assay, two substrates were used: as a first substrate the plasmid primer at different molar ratios. A final volume (50 µl) in buffer pUC19 DNA was digested with EcoRI and the 3«-end was C (50 mM Bistris, pH 6.5}1 mM DTT}250 µg}ml BSA}6mM $ $ labelled with [ H]dATP (1500 c.p.m.}pmol) using the Klenow MgCl#) contained the following components: 10 µM[H]dTTP fragment of E. coli pol I. The reaction was arrested by heating for (1.5 Ci}mmol), 200 ng of S. pombe recombinant PCNA, 0.5 µg 5 min at 65 mC, slowly cooled to room temperature and loaded poly(dA)}oligo(dT) and 10 µl of chromatographic fraction. DNA onto a Sephadex G-50 column pre-equilibrated with 10 mM synthesis was carried out at 37 mC for 60 min and the reaction $ Tris}HCl, pH 8}1 mM EDTA to eliminate unincorporated [ H]- was stopped by the addition of cold 10% (w}v) trichloroacetic dATP. As a second substrate, we used the partially mismatched acid (TCA). Insoluble radioactive material was collected, washed $ template}primer poly(dA)}oligo(dT)–[ H]dC-3«end-labelled, and counted as described [21]. prepared as described [6]. RF-C-dependent pol assay. Yeast cultures and extract preparations The final reaction volume of 25 µl contained: 50 mM Tris}HCl The S. pombe mutant strains polδts1 and polδts3 and the isogenic (pH 7.5), 1 mM DTT, 0.2 mg}ml BSA, 10 mM MgCl#, 200 ng of wild-type strain SP808 have been described previously [14]. S. pombe recombinant PCNA, 450 ng of E. coli single-stranded DNA-binding protein (SSB), 1 mM ATP, 100 ng of singly primed Procedures used for the extraction and purification of pol $ activities from fission yeast cells were similar to those described M13 DNA, [ H]dTTP (5 Ci}mmol), 6 µM dCTP, 6 µM dATP, previously for budding yeast [20]. Briefly, S. pombe cells were 6 µM dGTP, 0.02 units of pol δ and 0.03–0.05 units of calf grown aerobically at 30 mC (wild type) or 25 mC (thermosensitive thymus RF-C. Reactions were incubated at 37 mC, stopped by mutants) in 8 liters of 1% (w}v) yeast extract enriched with 3% the addition of 1 ml of ice-cold 10% TCA and acid precipitable (w}v) glucose and 12.5 g}l adenine. At a concentration of radioactivity was determined. One unit of RF-C-dependent ( 5¬10 }ml, cells were harvested by centrifugation at 2500 g at activity corresponds to the incorporation of 1 nmol of dNMPs 4 mC for 10 min and frozen in liquid nitrogen. Frozen cells were into acid-precipitable material during 1 h at 37 mC. broken with glass beads (0.5 mm diameter; Sigma) in a Waring To measure processivity, the sizes of products synthesized with Blender.