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Synthesis 2-14 Nucleotides Upstream of C2A12(C2-3/T2)

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Synthesis 2-14 Nucleotides Upstream of C2A12(C2-3/T2) Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4023-4027, June 1985 Biochemistry Mouse DNA polymerase a-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A12(C2-3/T2) sequences on a minute virus of mice DNA template (primase consensus formula/telomere replication/linear eukaryotic chromosome/discontinuous DNA synthesis) EMANUEL A. FAUST*t, RANDY NAGY*, AND SCOTT K. DAVEY*t *Cancer Research Laboratory and tDepartment of Biochemistry, University of Western Ontario, London, Ontario, N6A 5B7, Canada Communicated by Robert L. Sinsheimer, February 22, 1985 ABSTRACT The distribution of termination and initiation simple viral DNA molecule contains self-complementary sites in a 5081-nucleotide minute virus of mice DNA template sequences, 115 and 206 nucleotides long, at its 3' and 5' being copied by a highly purified mouse DNA polymerase termini, respectively; these regions contain the cis-dominant a-DNA primase complex in the presence of GTP has been genetic elements that comprise the origin for viral DNA examined. The 3'-hydroxyl termini (17 in all) were clustered at replication (14, 15). During the late S or early G2 phase of the six sites that were located 2-14 nucleotides upstream of cell cycle, the viral genome is converted to a double-stranded C2A2C2, C2AC3, or C2A2T2 sequences. When either [a-32p], or replicative form (RF) DNA by cellular enzymes, possibly [y-32P]GTP was included in the DNA polymerase reaction including DNA polymerase a (16). Viral DNA replication mixtures, nascent DNA became radiolabeled. Analysis of the proceeds through linear monomeric and dimeric replicative 32P-labeled material following treatment of the DNA with intermediates (17). In the present study, specific hexanucle- tobacco acid pyrophosphatase, bacterial alkaline phosphatase, otide sequences in the viral genome were correlated with or ribonuclease T1 revealed the presence ofoligoribonucleotide primase initiation sites. This observation has led us to chains averaging 5-7 nucleotides long and beginning with 5' propose the existence of a primase consensus formula that we GTP residues. Eight presumptive DNA primase initiation sites have called 4i. Alternatively, these could be termination were located opposite C4 or C5 sequences 3-9 nucleotides signals, thus indirectly stimulating primase at these locations. upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNAjunctions were found MATERIALS AND METHODS 3-10 nucleotides downstream of DNA primase initiation sites. Materials. DNA polymerase a-primase was purified from The results indicate that hexanucleotides having the general Ehrlich ascites mouse tumor cells and MVM DNA was formula C2A1.2(C2.3/T2), herein referred to as 4r, are involved isolated from purified virus particles (strain MVMp) as in promoting termination of DNA synthesis and/or de novo described (12, 15). Restriction endonucleases, DNA poly- initiation of RNA-primed DNA chains by DNA polymerase merase I (Klenow fragment), tobacco acid pyrophosphatase c-primase. (TAP), and bacterial alkaline phosphatase (BAP) were from Bethesda Research Laboratories or Boehringer Mannheim. Highly purified preparations of the eukaryotic replicative Ribonuclease T1 was from Sankyo. Nucleotides were from enzyme DNA polymerase a contain a tightly bound oligori- Sigma except for guanosine 5'-triphosphate 3'-phosphate, bonucleotide polymerase that is generally referred to as which was from P-L Biochemicals. Radioactive nucleotides primase (1, 2). Primase is distinguished from RNA polymer- were from New England Nuclear except for [y-32P]GTP, ases by the fact that the length of the oligoribonucleotide which was from ICN. Polyethyleneimine-cellulose plates synthesized is restricted (4-10 nucleotides) and by its ability were from Machery & Nagel (Brinkmann). to synthesize mixed oligoribo-deoxyribonucleotide chains DNA Polymerase a-Primase Reactions. Standard reactions (3-5). Primase purified from Drosophila melanogaster em- were carried out in a total volume of 16 ,ul containing 50 mM bryos appears to comprise polypeptides of Mr 50,000 and Tris HCl (pH 7.5), 4 mM MgCl2, 6 mM KCl, 8 mM dithio- 60,000 that are tightly bound to a Mr 182,000 DNA polymer- threitol, 1.6 mM 2-mercaptoethanol (from enzyme addition), ase catalytic subunit (6). An apparently homogeneous 27% (vol/vol) glycerol, bovine serum albumin at 400 ,g/ml, primase preparation from mouse hybridoma cells, free of 16 ,uM (each) dATP, dGTP, dCTP, dTTP, either 0.1 mM, 0.2 DNA polymerase activity, consists exclusively of polypep- mM, or 1.0 mM GTP, 10 ng of MVM DNA, 0.5 ,4Ci of tides of Mr 46,000 and 56,000 (7). Immunological evidence [a-32P]dTTP, and 1.0 unit of DNA polymerase a-primase. has been obtained for a tight association of primase activity Incubations were at 37°C for 2 hr and reactions were with DNA polymerase a from (murine) Ehrlich ascites cells terminated by the addition of 1/5th vol of agarose gel (8) and from KB cells (9). These physical and enzymological electrophoresis sample buffer (50 mM EDTA/2 M urea/2% properties of DNA polymerase a-primase are entirely con- NaDodSO4/1 M sucrose/0.1% bromophenol blue). sistent with a role for this enzyme in the discontinuous For the isolation of radiolabeled MVM DNA, standard RNA-primed DNA synthesis that occurs predominantly, but reaction mixtures were scaled up 20-fold and 25 uCi of not exclusively, on the lagging strand of the DNA replication [a-32P]dATP and 40 ,Ci of [a-32PJdGTP was added. Radio- fork in mammalian cells (10, 11). labeled primase products were prepared by scaling up stan- The ability of highly purified preparations of mouse DNA dard reaction mixtures 40-fold and adding 0.6 mCi (0.1 mM) polymerase a to copy the 5081-nucleotide linear single- of either [a-32p]- or [y-32P]GTP. Preparative-scale reaction stranded DNA genome of minute virus of mice (MVM), an mixtures were prewarmed (37°C, 5 min) prior to addition of autonomous parvovirus, has been described (12, 13). This DNA polymerase a-primase. Samples were adjusted to 5 mM The publication costs of this article were defrayed in part by page charge Abbreviations: MVM, minute virus of mice; RF, replicative form; payment. This article must therefore be hereby marked "advertisement" TAP, tobacco acid pyrophosphatase; BAP, bacterial alkaline phos- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phatase; kb, kilobase(s). 4023 Downloaded by guest on September 24, 2021 4024 Biochemistry: Faust et al. Proc. Natl. Acad. Sci. USA 82 (1985) EDTA, diluted with 1 vol of doubly distilled H20 or 10 mM Termination and Reinitiation at Preferred Sites on MVM Tris-HCl, pH 7.5/0.1 mM EDTA (TE buffer), and extracted DNA. When MVM single-stranded DNA was incubated with once with phenol (equilibrated with Tris HCl, pH 8.0) and DNA polymerase a-primase in the presence ofeither GTP or once with diethyl ether. The DNA was precipitated at -30'C the four rNTPs, the entire template was copied, resulting in in the presence of70o ethanol/0.3 M sodium acetate, pH 5.5, a DNA product that comigrated with authentic 5-kilobase- and tRNA (100 ktg/ml), dissolved in TE buffer, and further pair RF DNA in agarose gels run under nondenaturing purified by gel filtration on Sephadex G-50 (7.5-ml bed conditions. However, under alkaline conditions, major dis- volume). crete DNA fragments of "-9.0, 7.7, 7.5, and 2.1 kilobases (kb) Enzyme Digestion. Restriction enzymes (2 units per 20-plI were observed when GTP was included in reaction mixtures reaction mixture) were used according to the specifications (Fig. 2), and additional minor fragments of 1.1 and 0.9 kb provided by the supplier. Prior to treatment with either TAP, were also present (data not shown). The -9.0-kb DNA BAP, or ribonuclease T1, RF DNA samples were denatured species was present in samples prepared in the presence of at 960C for 3 min. TAP digestion (2 units, 370C, 60 min) was 0.1 mM GTP but absent in those prepared in the presence of carried out in 50 mM sodium acetate, pH 5.5/10 mM 1.0 or 4.0 mM GTP. At relatively low GTP concentrations 2-mercaptoethanol/1 mM EDTA. BAP treatment (70 units, (0.01 mM), a single 10-kb DNA species was observed (data 450C, 10 min) was carried out in 50 mM Tris HCl, pH 8.0. not shown). Fragments of -7.7, 7.5, 5.8, 5.6, and 2.1 kb, as well as DNA fragments ranging from 0.5 to i.5 kb, were seen Ribonuclease T1 digestion (5 units, 370C, 30 min) was carried when reactions were carried out in the presence of the four out in 50 mM Tris HCl, pH 8.0. rNTPs (Fig. 2). These data are consistent with the formation Polyacrylamide Gel Electrophoresis. Polyacrylamide slab of partially completed hairpin molecules resulting from ter- gels (0.4 mm x 40 cm) contained 8% acrylamide (acrylam- mination of DNA synthesis =0.6, 0.8, 2.5, 2.7, and 4.0 kb ide/bisacrylamide/19:1), 8 M urea, and 0.1 M Tris borate (pH from the 3' hairpin terminus of the 5.0-kb template strand. 8.3). The mixture was degassed and polymerized by the The absence of a 10.0-kb DNA species (even in overexposed addition of ammonium persulphate and N,N,N1,N'- autoradiograms) suggests that the frequency oftermination at tetramethylethylenediammine. tlectrophoresis was carried these sites approaches 100%. Inspection of the DNA se- out at 1.7 kV for 1.5-3 hr in 0.1 M Tris borate (pH 8.3). Gels quence at these locations (± 100 nucleotides) revealed the were exposed to x-ray film (Kodak XAR-5) for 1-10 days at presence of multiple closely related hexanucleotide se- -70'C with an intensifying screen.
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