The bacteriophage λ O protein binds to the λ replication origin (oriλ) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to oriλ is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production of crystals of the origin-binding domain of λ O that diffract to 2.5 Å is reported. Anomalous dispersion methods will be used to solve this structure.
Replication of single-stranded (SS) phage DNA proceeds by two mechanisms: discontinuous synthesis as observed with complementary-strand (−) synthesis (SS[+]→RF), where RF refers to replicative form, and continuous synthesis as observed with viral-strand (+) synthesis (RF→SS[+]). This paper deals with discontinuous synthesis and Eisenberg et al. (this volume) discusses continuous synthesis.
It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage λ O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of λ O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of λ O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with λ O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.
We have used a set of bacteriophage λ andEscherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of λ replication forks. We demonstrate that the λ replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650–750 nucleotides/s at 30 °C. This rate is identical to the fork movement rate we obtained using a minimal protein system, composed solely of E. coli DnaB helicase and DNA polymerase III holoenzyme. Our data are consistent with the conclusion that these two key bacterial replication proteins constitute the basic functional unit of a λ replication fork. A comparison of rolling circle DNA replication in the minimal and λ replication systems indicated that DNA synthesis proceeded for more extensive periods in the λ system and produced longer DNA chains, which averaged nearly 200 kilobases in length. The higher potency of the λ replication system is believed to result from its capacity to mediate efficient reloading of DnaB helicase onto rolling circle replication products, thereby permitting reinitiation of DNA chain elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually stimulated rolling circle DNA replication, but they apparently act indirectly by blocking accumulation of inhibitory free single-stranded DNA product. Finally, in the course of this work, we discovered thatE. coli DNA polymerase III holoenzyme is itself capable of carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. colisingle-stranded DNA-binding protein. We have used a set of bacteriophage λ andEscherichia coli replication proteins to establish rolling circle DNA replication in vitro to permit characterization of the functional properties of λ replication forks. We demonstrate that the λ replication fork assembly synthesizes leading strand DNA chains at a physiological rate of 650–750 nucleotides/s at 30 °C. This rate is identical to the fork movement rate we obtained using a minimal protein system, composed solely of E. coli DnaB helicase and DNA polymerase III holoenzyme. Our data are consistent with the conclusion that these two key bacterial replication proteins constitute the basic functional unit of a λ replication fork. A comparison of rolling circle DNA replication in the minimal and λ replication systems indicated that DNA synthesis proceeded for more extensive periods in the λ system and produced longer DNA chains, which averaged nearly 200 kilobases in length. The higher potency of the λ replication system is believed to result from its capacity to mediate efficient reloading of DnaB helicase onto rolling circle replication products, thereby permitting reinitiation of DNA chain elongation following spontaneous termination events. E. coli single-stranded DNA-binding protein and primase individually stimulated rolling circle DNA replication, but they apparently act indirectly by blocking accumulation of inhibitory free single-stranded DNA product. Finally, in the course of this work, we discovered thatE. coli DNA polymerase III holoenzyme is itself capable of carrying out significant strand displacement DNA synthesis at about 50 nucleotides/s when it is supplemented with E. colisingle-stranded DNA-binding protein. Investigations of the biochemical mechanisms involved in the initiation of bacteriophage λ DNA replication have been aided by the reconstitution of the initiation reaction with a defined set of purified λ and Escherichia coli proteins (1Mensa-Wilmot K. Seaby R. Alfano C. Wold M.C. Gomes B. McMacken R. J. Biol. Chem. 1989; 264: 2853-2861Abstract Full Text PDF PubMed Google Scholar, 2Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (203) Google Scholar). These studies have demonstrated that an ordered series of nucleoprotein structures are assembled at oriλ, the viral replication origin, and subsequently partially disassembled during the establishment of the apparatus responsible for replication fork propagation (2Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (203) Google Scholar, 3Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10699-10708Abstract Full Text PDF PubMed Google Scholar, 4Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar, 5Dodson M. McMacken R. Echols H. J. Biol. Chem. 1989; 264: 10719-10725Abstract Full Text PDF PubMed Google Scholar). The first step in the initiation pathway consists of the binding of multiple copies of the λ O protein to the viral origin and the subsequent self-assembly of this replication initiator into a nucleoprotein structure called the O-some (6Tsurimoto T. Matsubara K. Nucleic Acids Res. 1981; 9: 1789-1799Crossref PubMed Scopus (57) Google Scholar, 7Dodson M. Roberts J. McMacken R. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4678-4682Crossref PubMed Scopus (72) Google Scholar). The O-some both localizes the origin and serves as the foundation for the assembly of more complex nucleoprotein structures. Simultaneously, in solution, several molecules of the λ P protein bind to the hexameric E. coli DnaB helicase, to form a P·DnaB complex (8Wickner S.H. Cold Spring Harbor Symp. Quant. Biol. 1979; 43: 303-310Crossref PubMed Google Scholar, 9Klein A. Lanka E. Schuster H. Eur. J. Biochem. 1980; 105: 1-6Crossref PubMed Scopus (26) Google Scholar, 10Mallory J.B. Alfano C. McMacken R. J. Biol. Chem. 1990; 265: 13297-13307Abstract Full Text PDF PubMed Google Scholar). One or more molecules of this latter complex binds to the O-some, as a consequence of specific protein-protein interactions between O and P, to form a second stage nucleoprotein structure containing O, P, and DnaB. Protein-protein interactions within thisoriλ·O·P·DnaB structure prevent release and activation of the helicase activity of the DnaB molecule or molecules present in the complex (7Dodson M. Roberts J. McMacken R. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4678-4682Crossref PubMed Scopus (72) Google Scholar). Binding of the E. coli DnaJ protein to the oriλ·O·P·DnaB preinitiation structure (3Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10699-10708Abstract Full Text PDF PubMed Google Scholar) sets the stage for the ATP-dependent partial disassembly of the complex by the host DnaK (Hsp70) molecular chaperone (4Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar, 5Dodson M. McMacken R. Echols H. J. Biol. Chem. 1989; 264: 10719-10725Abstract Full Text PDF PubMed Google Scholar, 11Liberek K. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6632-6636Crossref PubMed Scopus (138) Google Scholar), a reaction that is aided by the GrpE protein (2Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (203) Google Scholar, 4Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar). This protein turnover reaction results in the release of a portion of the bound P and DnaJ proteins, as well as the liberation of DnaB and activation of its intrinsic helicase activity (4Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar, 5Dodson M. McMacken R. Echols H. J. Biol. Chem. 1989; 264: 10719-10725Abstract Full Text PDF PubMed Google Scholar, 11Liberek K. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6632-6636Crossref PubMed Scopus (138) Google Scholar, 12Dodson M. Echols H. Wickner S. Alfano C. Mensa-Wilmot K. Gomes B. LeBowitz J. Roberts J.D. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7638-7642Crossref PubMed Scopus (94) Google Scholar). If theoriλ template DNA is sufficiently negatively supercoiled, the released DnaB is inserted between the two DNA strands, presumably at the A + T-rich region of oriλ (7Dodson M. Roberts J. McMacken R. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4678-4682Crossref PubMed Scopus (72) Google Scholar, 12Dodson M. Echols H. Wickner S. Alfano C. Mensa-Wilmot K. Gomes B. LeBowitz J. Roberts J.D. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7638-7642Crossref PubMed Scopus (94) Google Scholar, 13Schnos M. Zahn K. Inman R.B. Blattner F.R. Cell. 1988; 52: 385-395Abstract Full Text PDF PubMed Scopus (90) Google Scholar), and becomes active as a replicative helicase. The transfer of DnaB helicase onto the DNA template completes the initiation phase of λ DNA replication and triggers a second series of protein-protein and protein-DNA interactions that culminate in assembly of the replication fork apparatus. The precise assembly pathway remains to be defined, but it is believed to include the following course of events. Single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; AMP-PNP, 5′-adenylylimidodiphosphate; bp, base pair(s); kb, kilobase(s); SSB, E. coli single-stranded DNA-binding protein; DNA pol IIIh, E. coli DNA polymerase III holoenzyme; RCR, rolling circle replication; TNC DNA, tailed, nicked circular DNA; BSA, bovine serum albumin. created by DnaB helicase action is stabilized by the binding of stoichiometric quantities of the E. coli single-stranded-DNA binding protein (SSB) (3Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10699-10708Abstract Full Text PDF PubMed Google Scholar, 12Dodson M. Echols H. Wickner S. Alfano C. Mensa-Wilmot K. Gomes B. LeBowitz J. Roberts J.D. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7638-7642Crossref PubMed Scopus (94) Google Scholar, 14LeBowitz J.H. McMacken R. J. Biol. Chem. 1986; 261: 4738-4748Abstract Full Text PDF PubMed Google Scholar). The E. coli primase (DnaG protein) binds transiently to the DnaB helicase (15McMacken R. Ueda K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4190-4194Crossref PubMed Scopus (63) Google Scholar, 16Tougu K. Marians K.J. J. Biol. Chem. 1996; 271: 21391-21397Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and subsequently synthesizes short RNA chains that serve as primers for synthesis of the leading and lagging DNA strands by the DNA polymerase III holoenzyme (DNA pol IIIh) (3Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10699-10708Abstract Full Text PDF PubMed Google Scholar, 15McMacken R. Ueda K. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4190-4194Crossref PubMed Scopus (63) Google Scholar). Biochemical studies of λ DNA replication in vitro have demonstrated that detectable levels of O, P, and DnaK remain associated with the oriλ template DNA following the chaperone-mediated partial disassembly reactions that bring about initiation of λ DNA replication (4Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar, 11Liberek K. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6632-6636Crossref PubMed Scopus (138) Google Scholar, 17Osipiuk J. Georgopoulos C. Zylicz M. J. Biol. Chem. 1993; 268: 4821-4827Abstract Full Text PDF PubMed Google Scholar). It is still uncertain if one or more of these proteins is associated with the λ replication fork apparatus. Although none of the evidence available to date suggests a direct role for the λ O and P replication proteins or the DnaK/DnaJ/GrpE chaperone system in the propagation of replication forks during λ DNA replication (4Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar), there remains a possibility that one or more of these proteins that act during the initiation phase of the reaction have an auxiliary role during the fork movement phase. We wished, therefore, to characterize the properties of replication forks established by the λ O and P proteins for comparison to the behavior of replication forks established with E. colireplication proteins. To study replication fork assembly and movement, we adapted the rolling circle replication assay pioneered by Lechner and Richardson (18Lechner R.L. Richardson C.C. J. Biol. Chem. 1983; 258: 11185-11196Abstract Full Text PDF PubMed Google Scholar), in which a tailed, nicked circular (TNC) DNA molecule serves as the replication template. With this assay, we can take advantage of the capacity of the λ O and P proteins to mediate the transfer of DnaB helicase onto any ssDNA, even if the ssDNA is stoichiometrically coated with SSB (19LeBowitz J.H. McMacken R. Nucleic Acids Res. 1984; 12: 3069-3088Crossref PubMed Scopus (16) Google Scholar, 20LeBowitz J.H. Zylicz M. Georgopoulos C. McMacken R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3988-3992Crossref PubMed Scopus (34) Google Scholar). A primary benefit of the rolling circle replication system for studies of the propagation of λ replication forks is that there are no topological constraints to encumber fork movement. This is in sharp contrast to the situation encountered with the standard oriλ plasmid replication assay, in which the rate of fork movement is probably limited by the rate at which topoisomerases relax the positive supercoils generated during replication of a covalently closed, circular template (21Baker T.A. Funnell B.E. Kornberg A. J. Biol. Chem. 1987; 262: 6877-6885Abstract Full Text PDF PubMed Google Scholar,22Hiasa H. Marians K.J. J. Biol. Chem. 1996; 271: 21529-21535Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Our studies demonstrate that the λ O and P replication proteins efficiently mediate the formation of robust replication fork assemblies. Such “λ replication forks” synthesize greater than 100 kb of DNA at rates approaching 750 nucleotides/s at 30 °C. This rate is indistinguishable from the rate of movement of replication forks established by a minimal two-protein system composed of DnaB helicase and DNA pol IIIh. The sources of materials were as follows: AMP-PNP, Sigma; Hepes, Research Organics; bovine serum albumin (BSA; fraction V) Miles Laboratory Inc.; unlabeled ribonucleoside triphosphates (rNTPs), unlabeled deoxyribonucleoside triphosphates (dNTPs), dT200 oligonucleotide and DEAE-Sephacel, Pharmacia Biotech Inc.; [methyl-3H]dTTP (40–70 Ci/mmol), ICN; [α-32P]dCTP (∼800 Ci/mmol) and [γ-32P]ATP (>5000 Ci/mmol), Amersham Corp. The buffers used were as follows: TE buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA); core buffer (10 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 100 μg/ml bovine serum albumin); high salt gradient buffer (50 mm Tris-HCl, pH 8.0, 0.7 mNaCl, 1 mm EDTA); neutral agarose-gel sample buffer (0.5% bromphenol blue, 60% (w/v) sucrose, 0.5% (SDS)); alkaline gel sample buffer (0.5% bromcresol green, 0.25 m NaOH, 10 mm EDTA, 0.5% SDS, 60% sucrose, and32P-labeled, linear pEMBL130(+) plasmid DNA (750 cpm/gel sample)); φX EDB (25 mm Tris-HCl, pH 7.5, 10 mm dithiothreitol, 100 μg/ml BSA, 5% sucrose); buffer A (50 mm Tris-HCl, pH 7.5, 20% (v/v) glycerol, 1 mm EDTA, 1 mm dithiothreitol, 5 mmMgCl2, 0.1 m NaCl); TGE buffer (25 mm Tris, 0.19 m glycine, 1 mmEDTA); and S1 nuclease buffer (50 mm sodium acetate, pH 4.8, 0.5 m NaCl, 1 mm ZnCl2, 30 μg/ml denatured calf thymus DNA). The E. coli K-12 strains used and their relevant genetic characteristics were as follows. SK6776 (uvrD288) is a DNA helicase II mutant (provided by Dr. C. McHenry, University of Colorado School of Medicine); C600dnaK103/pJK23 (23Lipinska B. King J. Ang D. Georgopoulos C. Nucleic Acids Res. 1988; 15: 7545-7562Crossref Scopus (53) Google Scholar) is a GrpE protein overproducer (provided by Dr. C. Georgopoulos, University of Geneva); 71-18 (F′) and 71-18/pEMBL130(+) (24Dente L. Cortese R. Methods Enzymol. 1987; 155: 111-119Crossref PubMed Scopus (54) Google Scholar) were used for propagation of M13 phage derivatives (provided by Dr. G. Cesareni, University of Tor Vergata (Rome)); RLM727 (HfrH/pRLM55) is a thermoinducible strain for amplification of E. coli SSB that was constructed in this laboratory (25.LeBowitz, J. (1985) Biochemical Mechanisms of Strand Initiation in Bacteriophage λ DNA Replication. Ph.D. dissertation, Johns Hopkins University, Baltimore.Google Scholar); RLM861 (N100/pRLM31) is arecA −, thermoinducible, DnaB-overproducing strain that was constructed in this laboratory. 2C. Loehrlein and R. McMacken, unpublished data. Strain RLM973, which is 71-18/pRLM85, is described in this paper. λ DNA and λ/HindIII DNA size standards were purchased from New England Biolabs, Inc.; T7 DNA was purchased from the U.S. Biochemical Corp. Phage M13mp19 was a gift of Drs. T. Seeley and L. Grossman of this department. λ/KpnI and linear pEMBL130(+) DNA size standards were prepared by digesting these DNAs with KpnI and BamHI, respectively, followed by treatment with calf intestine phosphatase. Protein was removed by phenol extraction, and the DNAs were concentrated by ethanol precipitation. The plasmid pRLM85 was constructed by E. Hwang in this laboratory by cloning a Sau3AI fragment of λ DNA (positions 38815–39577; Ref. 26Sanger F. Coulson A.R. Hong G.F. Hill D.F. Petersen G.B. J. Mol. Biol. 1982; 162: 729-773Crossref PubMed Scopus (869) Google Scholar), containing the λ origin of DNA replication, into the BamHI site of pEMBL130(+) such that the R (lower) strand of oriλ is inserted into the (+)-strand of the plasmid. M13mp19oriλL was constructed by cloning from pRLM85 a 0.8-kb PstI to SacI fragment, containing oriλ and the majority of the pEMBL130(+) polylinker sequence, into M13mp19 DNA that had been digested with both PstI and SacI, which removed most of the mp19 polylinker sequence. Phage M13K07 (27Vieira J. Messing J. Methods Enzymol. 1987; 153: 3-11Crossref PubMed Scopus (2007) Google Scholar) was a gift of Drs. E. Hildebrand and L. Grossman of this department. Oligodeoxyribonucleotides were constructed by automated solid phase synthesis by S. Morrow of this department and were used without further purification. Oligonucleotide RM34 is 5′-CGGACCTGCAGGCAT-3′; it is complementary to (+)-strand DNA at the position of the unique PstI site of M13mp19oriλL. Oligonucleotide RM35 is 5′-AATTCGAGCTCGATAT-3′; it is complementary to (+)-strand DNA at the position of the unique SacI site of M13mp19oriλL. PstI,SacI, KpnI, and BamHI restriction endonucleases and T4 polynucleotide kinase were from New England Biolabs. Calf intestine phosphatase, proteinase K, and DNase I were from Boehringer Mannheim. S1 nuclease was from Pharmacia. HomogeneousE. coli UvrD protein (15,000 units/mol) (28Runyon G.T. Lohman T.M. J. Biol. Chem. 1989; 264: 17502-17512Abstract Full Text PDF PubMed Google Scholar) was a generous gift of Drs. Jaya Yodh and Randy Bryant of this department. Antibody directed against E. coli UvrD protein was graciously donated by Drs. Robert Lahue and Paul Modrich (Duke University). The β-subunit of the E. coli DNA pol IIIh was kindly provided by Dr. Mike O'Donnell (Rockefeller University). All λ and E. coli replication proteins used were estimated to be greater than 95% pure. Purification and specific activities of the λ O and P replication proteins and the E. coli DnaJ, DnaK, DnaG primase, and DNA polymerase III holoenzyme proteins have been described previously (1Mensa-Wilmot K. Seaby R. Alfano C. Wold M.C. Gomes B. McMacken R. J. Biol. Chem. 1989; 264: 2853-2861Abstract Full Text PDF PubMed Google Scholar). Some preparations of DNA pol IIIh (3 × 105 units/mg) were purified from SK6776 (uvrD) essentially as described (29McHenry C. Kornberg A. J. Biol. Chem. 1977; 252: 6478-6484Abstract Full Text PDF PubMed Google Scholar). DNA polymerase III* was purified by a modification of the published protocol (30Maki H. Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6570-6578Abstract Full Text PDF PubMed Google Scholar), using a procedure developed by Dr. M. O'Donnell (Rockefeller University).E. coli SSB (7 × 104 units/mg) was purified from strain RLM727 (HfrH/pRLM55) by a modification of the protocol of LeBowitz (25.LeBowitz, J. (1985) Biochemical Mechanisms of Strand Initiation in Bacteriophage λ DNA Replication. Ph.D. dissertation, Johns Hopkins University, Baltimore.Google Scholar). Briefly, the blue dextran-Sepharose column was replaced by a ssDNA cellulose column prepared as described by Alberts (31Alberts B.M. Amodio F.J. Jenkins M. Gutmann E.D. Ferris F.L. Cold Spring Harbor Symp. Quant. Biol. 1968; 33: 289-305Crossref PubMed Scopus (254) Google Scholar). Approximately 57.5 mg of fraction II protein (>95% SSB) was diluted with buffer A (minus NaCl) to a conductivity equivalent to buffer A and applied at 3 column volumes/h to an ssDNA cellulose column (150 ml, 5.75 mg ssDNA/ml) that had been equilibrated with buffer A. The column was washed with buffer A containing 0.25 m NaCl until the absorbance at 280 nm approached zero. Bound SSB was eluted with buffer A containing 2 m NaCl and purified further by chromatography on hydroxyapatite as described (25.LeBowitz, J. (1985) Biochemical Mechanisms of Strand Initiation in Bacteriophage λ DNA Replication. Ph.D. dissertation, Johns Hopkins University, Baltimore.Google Scholar). E. coliGrpE protein (6.4 × 105 units/mg) was purified essentially as described by Zylicz et al. (32Zylicz M. Ang D. Georgopoulos C. J. Biol. Chem. 1987; 262: 17437-17442Abstract Full Text PDF PubMed Google Scholar), except that the strain used was C600dnaK103/pJK23 (23Lipinska B. King J. Ang D. Georgopoulos C. Nucleic Acids Res. 1988; 15: 7545-7562Crossref Scopus (53) Google Scholar). E. coli DnaB protein was purified from 300 g of thermally induced RLM861 by a modification2 of the protocol of Ueda et al. (33Ueda K. McMacken R. Kornberg A. J. Biol. Chem. 1978; 253: 261-269Abstract Full Text PDF PubMed Google Scholar). Following the 0.24 ammonium sulfate and two 0.20 ammonium sulfate backwashes of the 0–40% ammonium sulfate precipitate, the residual protein precipitate was resuspended in 18 ml of buffer A (fraction II, 27 ml, 1070 mg, 4.7 × 107units). A 4-ml portion of fraction II (160 mg of protein) was diluted 3-fold with buffer A and applied to a 75-ml ATP-Sepharose column that had been prepared by the technique of Lamed (34Lamed R. Levin Y. Oplatka A. Biochim. Biophys. Acta. 1973; 305: 163-171Crossref PubMed Scopus (63) Google Scholar), except that the amount of adipic dihydrazide used as a linker ligand was reduced 10-fold to 0.9 g/100 ml. This modification resulted in lowered amounts of ATP covalently linked to the Sepharose matrix (typically 1 μmol of ATP/ml of gel matrix), a reduction that correlated with improved stability of DnaB protein eluted from the ATP affinity column. 3K. Stephens and R. McMacken, unpublished data. The ATP-Sepharose column was washed with 5 column volumes of buffer A, followed by 10 column volumes of buffer A containing 10 mm MgCl2 and 10 mm AMP. DnaB protein was subsequently eluted with buffer A with 20 mm sodium pyrophosphate (fraction III, 11 mg, 2.3 × 106 units). Fraction III was applied to a 7-ml DEAE-Sephacel column that had been equilibrated with buffer A. The column was washed with 5 volumes of buffer A containing 0.15m NaCl, followed by 2 column volumes of buffer A containing 0.2 m NaCl. DnaB was eluted with 5 column volumes of buffer A containing 0.4 m NaCl (fraction IV, 6 mg, 3.4 × 106 units). The plasmid pEMBL130(+) was propagated in the ssDNA form by superinfection of 71-18/pEMBL130(+) cells with the helper phage M13K07 by the method of Dente et al. (27Vieira J. Messing J. Methods Enzymol. 1987; 153: 3-11Crossref PubMed Scopus (2007) Google Scholar). M13mp19oriλL was propagated in the E. colistrain 71-18; phage particles were isolated from the cell-free supernatant as described (35Yamamoto K.R. Alberts B.M. Benzinger R. Lawhorne L. Treiber G. Virology. 1970; 40: 734-744Crossref PubMed Scopus (968) Google Scholar). Phage DNA was isolated free of contaminating protein and RNA by using a modification of the plasmid isolation protocol of Birnboim and Doly (36Birnboim H.C. Doly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9908) Google Scholar). Phage prepared from a 1-liter culture of infected E. coli, as described above, were resuspended in 5 ml of TE buffer and denatured by the addition of 10 ml of a solution containing 0.2 m NaOH and 1% SDS. Contaminating protein and RNA was precipitated by the addition of 7.5 ml 3 m sodium acetate, pH 4.8. Following centrifugation, the DNA remaining in the supernatant was precipitated by an equal volume of ethanol. The pEMBL130(+) ssDNA prepared by this technique is suitable for digestion with restriction enzymes. For some preparations of the tailed rolling circle DNA template, the circular single-stranded pEMBL130(+) plasmid DNA was isolated free of helper phage DNA by sucrose gradient centrifugation. The DNA mixture was diluted with high salt gradient buffer and layered onto an 11-ml 5–30% sucrose gradient prepared in high salt gradient buffer (each tube contained a 1-ml cushion of 50% sucrose). The DNA samples were centrifuged in a Beckman SW41 rotor at 25,000 rpm for 16 h at 20 °C. Fractions (0.5 ml) were collected from the bottom, and portions of each fraction were analyzed for the presence of DNA by neutral agarose gel electrophoresis. The fractions free of helper phage DNA were pooled, and the pEMBL130(+) DNA was concentrated by ethanol precipitation. The DNA sample (350 μg) was resuspended in 0.5 ml of TE buffer, adjusted to 0.1 m NaOH and applied to a 12-ml Bio-Gel A-15 m column that had been equilibrated with 0.1 mm Tris base. Fractions (0.5 ml) were collected and subsequently analyzed by neutral agarose gel electrophoresis. The fractions containing the peak of pEMBL130(+) ssDNA were pooled, neutralized by the addition of 119 volume of 1 mTris-HCl, pH 8, and precipitated with ethanol. The precipitate was collected by centrifugation and resuspended in TE buffer, and the purified circular ssDNA was used for the preparation of tailed DNA circles. The ssDNA 800-nucleotide primer (800-mer) used to form the 5′-tail of the TNC DNA template and helicase substrate was prepared by digesting ssDNA at restriction sites made duplex by the presence of hybridized oligonucleotides. 500 μg of mp19oriλL ssDNA was suspended in 3.2 ml of core buffer and mixed with 42.3 μg of oligonucleotide RM34, 17.3 μg of oligonucleotide RM35,PstI (600 units), and SacI (800 units); the mixture was incubated at 37 °C for 60 min. The 800-mer was isolated free of linear M13mp19 DNA by neutral agarose gel electrophoresis and was recovered from the gel by electroelution using a Schleicher and Schuell Elutrap device. Some preparations of the 800-mer were treated with calf intestine phosphatase before purification by gel electrophoresis. This permits efficient end labeling of the linear strand of the helicase substrate/rolling circle DNA template by T4 polynucleotide kinase for preparation of a labeled size marker. Hybridization of the 800-mer to the pEMBL130(+) ssDNA (the 800-mer contains at its 3′ terminus a 42-nucleotide-long sequence that is complementary to the pEMBL130(+) polylinker) was accomplished by mixing 250 μg of circular ssDNA with 25 μg of the 800-mer in TE buffer containing 0.5 m NaCl in a final volume of 1.25 ml. The mixture was incubated at 65 °C for 2 min and then immediately supplemented with 1.37 mg of E. coli SSB and incubated for an additional 60 min at 37 °C. The final DNA concentration was 0.14 mg/ml and the molar ratio of pEMBL130(+) ssDNA to 800-mer was 2:1. The hybrid was diluted 7.67-fold into φX EDB containing 2.5 mm ATP, 13 mm MgCl2, 2.5 mm spermidine chloride, 100 μm each dATP, dCTP, dGTP, and dTTP, 16 μg of DNA pol IIIh β-subunit, 81 μg of SSB, and 67,500 units of DNA polymerase III* (φX EDB replication mix). Some template batches were prepared using partially purified DNA pol IIIh ∼(2 × 105 units/mg; Ref. 29McHenry C. Kornberg A. J. Biol. Chem. 1977; 252: 6478-6484Abstract Full Text PDF PubMed Google Scholar) instead of DNA polymerase III* and β-subunit. DNA synthesis was carried out during a 30-min incubation at 30 °C and was terminated by chilling the mixture to 0 °C. In some instances, radiolabeled TNC DNA template was prepared. On these occasions, the replication mixture contained [3H]dNTPs or [α-32P]dNTPs at approximately 100–200 cpm/pmol of dNTP. The TNC DNA product was concentrated 10-fold using an Amicon Centricon 30 device. Replication proteins and unreplicated pEMBL130(+) ssDNA were removed from the TNC DNA template by sucrose gradient centrifugation. The DNA sample was supplemented with SDS to 1% (w/v) and diluted with 0.25 volume of 5 × high salt gradient buffer. Portions were layered onto neutral 5–30% sucrose gradients and centrifuged and analyzed as described above. The fractions containing double-stranded DNA were pooled, and the TNC DNA product was concentrated by ethanol precipitation. For those instances in which helper phage DNA was first removed from pEMBL130(+) ssDNA by sucrose gradient centrifugation before synthesis of the TNC DNA template, a simplified protocol was used for synthesis and purification of the template. The 800-mer was mixed with pEMBL130(+) ssDNA at a molar ratio of 1.5 (800-mer:circle) and hybridized as described above. Once formed, the DNA hybrid was diluted into φX EDB replication mix as described above, except that the mix contained 100,000 units of DNA pol IIIh instead of DNA polymerase III* and β-subunit. The TNC DNA replication product prepared in this manner had negligible amounts of contaminating single-stranded DNA. The TNC DNA sample was incubated for 4 h at 55 °C with proteinase K (1 mg/ml in 0.5% N-lauroylsarcosine) and extracted three times in succession with neutralized phenol/chloroform/isoamyl alcohol (24:24:1). DNA product in the aqueous phase was precipitated with ethanol, resuspended in TE buffer, and used directly as a substrate/template in rolling circle DNA replication assays. The reaction
I "Replication of the chromosome of bacteriophage X depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host.As previously described, the first stage in this process is the binding of multiple copies of the X 0 initiator to the X replication origin (oriX) to form the nucleosomelike 0-some.The 0-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of X DNA replication to oriX.To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified.Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at oriX during the initiation of X DNA replication.A second-stage oriX-0 (X 0 protein)*P (X P protein)*DnaB nucleoprotein structure is formed when 0, P, and E. coli DnaB helicase are incubated with oriX DNA.In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an oriX-O*P*DnaB*DnaJ complex.Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of X DNA replication.The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, a n d i n a f o u r t h -s t a g e r e a c t i o n a complete oriX-0-P*DnaB*DnaJ*DnaK initiation complex is assembled.Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a par- tial disassembly of the fourth-stage complex.These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the oriX template.The protein diassembly reactions are described in the accompanying articles (
Cyclohexene nucleic acids (CeNA) contain a cyclohexene ring instead of the normal -D-2'-deoxyribose.The cyclohexene oligonucleotide GTGTACAC was synthesized using phosphoramidite chemistry and standard protecting groups [1].CeNA is stable against enzymatic degradation and induces RNaseH activity.CeNA also forms more stable duplexes with RNA than its natural analogues [2] [3].Crystals of GTGTACAC were obtained at 289K by the hangingdrop vapour-diffusion technique.The crystals diffract to 1.7 Å resolution and belong to the trigonal space group R3 with unit-cell parameters a = 41.434 and c = 66.735Å.The structure of a fully modified GTGTACAC sequence with left handed CeNA building blocks is presented.Particular interests concern the puckering of the sugar moiety, helical parameters and the hydration of the double helix.
Synthesis of the oligonucleotides that prime replication of phiX174 single-stranded DNA employs complex protein machinery of the host cell which is probably used by the cell to replicate its own chromosome. Primer synthesis depends on at least five proteins (DNA binding protein, dnaB and dnaC proteins, protein i, and protein n) and ATP to form a replication intermediate and another protein, primase (dnaG protein), to assemble the oligonucleotide by template transcription. The data in this paper show that ribo- and deoxyribonucleoside triphosphates can serve as substrates and form hybrid primers when present together. Both RNA and DNA primers were initiated with ATP. At least three of the four base-pairing nucleoside triphosphates were required for the transcription that generates effective primers. Over 90% of the RNA and DNA transcripts were extended into complementary strands by DNA polymerase III holoenzyme. At optimal triphosphate concentrations, the rate and extent of primer formation were greater from ribonucleoside triphosphates than from deoxyribonucleoside triphosphates. Uncoupled from DNA replication, the length of RNA primers was 14 to 50 residues, the DNA primers 4 to 20 residues. The fingerprint pattern of an RNase digest of RNA primers has a complexity suggestive of transcription from many sites on the phiX174 template. The multienzyme priming system is highly specific for phiX174 DNA as template.
Among the many viral, bacterial, and animal DNA polymerases isolated thus far none can start a chain in vitro (Kornberg 1974). In addition to a template strand, each requires a 3′-OH-terminated chain (primer terminus). Because they lack such termini, the coliphage circular, single-stranded (SS) DNA chromosomes have proved to be ideal templates for studying the mechanisms used by Escherichia coli to initiate new DNA chains (Schekman et al. 1974; Wickner and Hurwitz 1975a). Because RNA polymerases are known to initiate new RNA chains, it appeared that an RNA transcript might prime DNA synthesis (Brutlag et al. 1971), and rifampicin, a specific inhibitor of E. coli RNA polymerase, should therefore inhibit DNA synthesis. RNA polymerase was shown to be essential in the conversion of phage M13 SS DNA to the duplex replicative form (RF). Later it was demonstrated that soluble extracts from gently lysed E. coli cells support the conversion of various bacteriophage SS DNAs to their replicative forms (Wickner et al. 1972; Schekman et al. 1974). Fractionation and identification of the proteins involved in this apparently simple replication showed not only that synthesis of the complementary DNA chain is surprisingly complex, but also that there are at least three different DNA-strand initiation systems in E. coli (Schekman et al. 1974; Wickner and Hurwitz 1974; Schekman et al. 1975; Kornberg 1977). The chromosomes of the filamentous phage M13 and the isometric phages G4 and ϕ X174 are each replicated in vitro by a different set of bacterial replication proteins, which differ primarily...
The preptriming steps in the initiation of bactericphage λ DNA replication depend on the action of the λ O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the λ O protein to the phage replication origin (oriλ) initiates the ordered assembly of a series of nucleoprotein structures that form at oriλ prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of A DNA replication is known to occur only on supercoiled templates in vivo and in vitro , We examined how the early steps in A DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed oriλ DNA. Nonetheless, the DNA templates in these structures must be negatively super-twisted before they can be replicated. Once DNA helicase unwinding is initiated at oriλ, however, later steps in A DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of A DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.
