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DNA Or RNA Priming of Bacteriophage G4 DNA Synthesis By Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2815-2819, July 1977 Biochemistry DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein (ADP/DNA binding protein/bacteriophage ST-1 DNA/DNA replication) SUE WICKNER Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Jerard Hurwitz, May 6, 1977 ABSTRACT Escherichia coli dnaG protein is involved in wild-type and thermolabile) were purified by procedures in the initiation of DNA synthesis dependent on G4 or ST-1 sin- refs. 13, 14, 15, 16, 5, and 17, respectively. Assay conditions and gle-stranded phage DNAs [Bouche, J.-P., Zechel, K. & Kornberg, definitions of units for DNA polymerase III are in ref. A. (1975)1. Biol. Chem. 250,5995-60011. The reaction occurs by 3; those the following mechanism: dnaG protein binds to specific sites for the other proteins are in the above references. DNA EF I on the DNA in a reaction requiring E. coli DNA binding protein. and DNA EF III were purified by procedures to be published An oligonucleotide is synthesized in a reaction involving dnaG elsewhere using the assay described in ref. 4. By native poly- protein, DNA binding protein, and DNA. With G4 DNA this acrylamide gel electrophoresis (18), the DNA binding protein reaction requires ADP, dTTP (or UTP), and dGTP (or GTP). was 80% pure and the dnaG protein from wild-type cells was Elongation of the oligonucleotide can be catalyzed by DNA 30% polymerase II or III in combination with dnaZ protein and pure. The wild-type dnaG protein contained 3400 units/ DNA elongation factors I and III, presumably by the mecha- mg of protein; the thermolabile dnaG protein [purified from nism previously reported [Wickner, S. (1976) Proc. Natl. Acad. E. col strain NY73 (dnaG3)] contained 600 units/mg of pro- Sci. USA 73 3511-3515] or by DNA polymerase I. tein. A unit of dnaG protein catalyzes the incorporation of 1 nmol of dTMP in 20 min at 30' in the OX174 DNA-dependent Three pathways have been described by which single-stranded complementation assay (17). T4 DNA polymerase was the circular phage DNAs can be converted to double-stranded generous gift of N. Nossal. DNA in reactions catalyzed by purified Escherichia coli pro- teins (1-3). The three pathways use different mechanisms to RESULTS prime the template DNA but share a common mechanism of Requirements for G4 Phage DNA-Dependent DNA Syn- DNA elongation (4). In the elongation reaction, DNA elonga- thesis. DNA synthesis dependent on G4 and ST-1 DNA re- tion factor III (EF III) and dnaZ protein catalyze the transfer quired dnaG protein, DNA binding protein, DNA polymerase of DNA elongation factor I (EF I) to primed DNA in an ATP- III, dnaZ protein, DNA EF 1, and DNA EF III (Table 1A). (or dATP-) dependent reaction; DNA polymerase III binds to ADP stimulated the reaction; the Km was about 2 ,M. ADP the primed template-DNA EF I complex and catalyzes DNA could not be replaced by any other ribo or deoxyribonucleoside elongation (4). In the simplest pathway, fd (or M13) DNA diphosphates or triphosphates, AMP, or a,#-methylene ADP synthesis is primed by RNA synthesized by RNA polymerase (all at 20 ,uM). ATP and dADP stimulated DNA synthesis to the (1, 3,5). In the most complex pathway, OX174 DNA synthesis same extent but only at higher concentrations; their Km was requires dnaB, dnaC(D), and dnaG proteins, DNA binding about 50 ,uM. The amount of stimulation by ADP was greater protein, at least three other E. coli proteins [DNA replication at lower concentrations of dnaG protein. With reactions as factors X, Y, and Z (2)], only ATP of the rNTPs (2), and the same described in Table IA with G4 DNA and 0.02,0.4, and 4.0 unit four DNA elongation proteins (1, 2). Most likely, the first seven of dnaC protein, <0.1, 2.9, and 13.1 pmol of dTMP was in- proteins are involved in primer synthesis. In a third pathway, corporated, respectively, in the absence of ADP and 6.2, 32.8, G4 (ST-1, OXtB) DNA synthesis requires dnaG protein (6), and 33.0 pmol, respectively, in the presence of ADP. Varying DNA binding protein, and the DNA elongation proteins (5). the concentration of DNA binding protein or DNA had no ef- In this report, the mechanism of priming of G4 DNA synthesis fect on the amount of ADP stimulation. Similar results were is examined. obtained with ST-i DNA; however, at all concentrations of dnaG protein, DNA synthesis independent of ADP was greater MATERIALS AND METHODS than with G4 DNA. Materials and Methods. Unless otherwise indicated, these The requirements for G4 and ST-1 DNA synthesis by DNA were as described (7). rNTPs were chromatographed on polymerase II were similar to those with DNA polymerase III DEAE-Sephadex (8) and dNTPs were treated with 5 ,umol of (Table 1B). DNA polymerase I (Table 1C) and T4 DNA poly- NaIO4 per ,umol of nucleotide for 30 min at 25°. [a-32P]dNTPs merase (results not shown) also catalyzed G4 and ST-1 DNA were from New England Nuclear Corp. synthesis in reactions requiring dnaC protein, DNA binding Preparation of DNA. qX174 and fd DNA were prepared protein, and ADP; the reactions did not require the elongation as described (9, 10). G4 and ST-I phage were grown (11, 12), proteins. These results are consistent with the previous obser- banded twice in CsCl, and sedimented in 10-30% sucrose vation that DNA elongation of any primed single-stranded gradients containing 1 M NaCl/1 mM EDTA/30 mM Tris-HCl DNA by DNA polymerase II or III was stimulated by the (pH 8.0). The DNA was extracted with phenol (9). elongation components, while elongation by DNA polymerase Preparation of Proteins. DNA polymerases I, II, and III, I or T4 DNA polymerase was not significantly stimulated DNA binding protein, dnaZ protein, and dnaG protein (both (4). In contrast to G4 and ST-I DNA, fd and OX174 DNA were Abbreviations: DNA EF I, DNA elongation factor I; DNA EF III, DNA inactive for DNA synthesis by these proteins. With the condi- elongation factor III. tions described in Table 1, <0.1 pmol of dTMP was incorpo- 2815 2816 Biochemistry: Wickner Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Requirements for G4 and ST-1 DNA-dependent DNA synthesis 1.2 dTMP incorporated (pmol/20 min) 0.8 Additions G4 DNA ST-1 DNA 0.4 A. DNA polymerase III 0.8 Complete 25.2 25.0 -dnaG protein <0.1 <0.1 0.4 -DNA binding protein 0.3 0.3 -DNA EF I 0.X 0.6 -DNA EF III 0.6 0.2 -dnaZ protein 0.5 0.2 0.8IC -DNA polymerase III 0.4 0.4 -ADP 1.9 5.0 0.4 -ADP + other nucleotides* 1.7-3.1 - is B. DNA polymerase II D -E Complete 29.5 17.3 11 0.8 -dnaG protein 0.3 0.2 -DNA binding protein <0.1 0.4 r 0.4 -DNA EF I 3.2 0.4 20 -DNA EF III 2.8 3.4 0a -dnaZ protein 2.5 1.5 1.6 -DNA polymerase II 0.4 0.4 C 1 -ADP 1.9 7.5 1.2~ C. DNA polymerase I Complete 26.4 34.4 0.8 -dnaG protein <0.1 <0.1 -DNA binding protein 2.2 3.2 0.4 -DNA polymerase I <0.1 <0.1 -ADP 0.7 9.0 Reaction mixtures (30 ul) contained: reaction buffer (1 mM di- 0.8 thiothreitol/20 Mg of rifampicin per ml/50 mM Tris-HCl (pH 7.5)/1 mg of bovine serum albumin per ml/7 mM MgCl2), 50 MAM each of 0.4 dATP, dGTP, dCTP, and [3HJdTTP (800 cpm/pmol), 12 MM ADP, 300 pmol of DNA, 0.8 Mg of DNA binding protein, and 0.04 unit of dnaG protein. Mixtures for A and B contained 0.2 unit ofdnaZ pro- 0.8 G tein, 0.2 unit of DNA EF I, and 0.2 unit of DNA EF III. Mixtures for A, B, and C contained 0.3 unit ofDNA polymerase III, 0.3 unit ofDNA polymerase II, and 0.2 Mg of DNA polymerase I, respectively. A unit 0.4 of DNA polymerase catalyzes the incorporation of 1 nmol of dTMP in 30 min at 300 with DNase-treated salmon sperm DNA (3). After 4 8 12 16 20 24 28 3 15 min at 300, acid-insoluble radioactivity was measured. Fraction number * Nucleotides substituted for ADP at 20 MM were: all four rNTPs, three other rNDPs, four dNTPs, four dNDPs, AMP, and a,j#- FIG. 1. Site-specific binding of dnaG protein to G4 and ST-1 methylene-ADP. DNA. (A) The first reaction mixture (60 Ml) contained: reaction buffer as in Table 1, 6 nmol ofG4 DNA (as nucleotide), 8Mg of DNA binding protein, and 1.7 units of dnaG protein. After 10 min at 300, the mix- rated with fd DNA and 0.4-1.9 pmol was incorporated with ture was applied at room temperature to a 22 X 0.5-cm column of Sepharose 6B equilibrated with 50 mM Tris-HCl (pH 7.8)/1 mM di- 4X174 DNA. thiothreitol/0.1 mg of bovine serum albumin per ml/5% glycerol/5 mM Site-Specific Binding of dnaG Protein to G4 and ST-1 MgCl2.
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