dna replication

Preface The increasing relevance of studies of DNA replication and DNA repair to the understanding of human genetic disease, cancer, and aging is bringing growing numbers of investigators into this field. The rich legacy of past studies of the enzymology of these processes has already had wide impact on how modern biological research is conducted in that it provided the roots for the whole field of genetic engineering. The work of the biochemist in characterizing these complex reactions is still far from done, however, since we are still short of the mark of being able to use our knowledge to prevent the devastating aberrations caused by failures of faithful copying of the genome by the self-editing DNA replication and repair apparatus. Past study of the enzymes involved in DNA replication has given rise to a number of highly refined approaches to defining their individual enzymatic mechanisms and how they interact to carry out the process of DNA replica- tion in the cell. These methods form the foundation on which even more detailed understanding, driven and directed by the revolutionary addition of structural information on these proteins at the atomic level, will necessar- ily be built. This volume contains a series of articles by the main contributors to this field which form a guide to students of nucleic acid enzymology who wish to study these types of proteins at ever increasing levels of resolution. Descriptions of functional, structural, kinetic, and genetic methods in use for analyzing DNA polymerases of all types, viral reverse transcriptases, helicases, and primases are presented. In addition, a number of chapters describe strategies for studying the interactions of these proteins during replication, in particular recycling during discontinuous synthesis and cou- pling of leading and lagging strands. Comprehensive descriptions of uses of both prokaryotic and eukaryotic crude in vitro replication systems and reconstitution of such systems from purified proteins are provided. These chapters may also be useful to investigators who are studying other multien- zyme processes such as recombination, repair, and transcription, and begin- ning to study the coupling of these processes to DNA replication. Methods of analyzing DNA replication in vivo are also included. JUDITH L. CAMPBELL xiii Contributors to Volume 262 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. EDWARD ARNOLD (15), Center for Advanced Biotechnology and Medicine, and Chemis- try Department, Rutgers University, Piscata- way, New Jersey 08854-5638 ROBERT A. BAMBARA (21), Departments of Biochemistry, Microbiology and Immunol- ogy, and the Cancer Center, University of Rochester, Rochester, New York 14642 MARJORIE H. BARNES (4), Department of Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 BLAINE BARTHOLOMEW (37), Department of Medical Biochemistry, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-650.3 DANIEL W. BEAN (29), Department of Biol- ogy, University of North Carolina, Chapel Hill, North Carolina 27599 WILLIAM A. BEARD (11), Scaly Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555- 1068 KATARZyNA BEBENEK (18), Laboratory of Molecular Genetics, National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709 WILLIAM R. BEBRIN (24), Department of Bio- logical Chemistry and Molecular Pharma- cology, Harvard Medical School, Boston, Massachusetts 02115-5747 STEPHEN J. BENKOVlC (13, 20, 34), Depart- ment of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 ROLF BERNANDER (45), Department of Bio- physics, Institute for Cancer Research, The Norwegian Radium Hospital 0310 Oslo, Norway STACY BLAIN (27), Department of Biochemis- try and Molecular Biophysics, Howard ix Hughes Medical Institute, Columbia Uni- versity, College of Physicians and Surgeons', New York, New York 100.32 Luxs BLANCO (5, 22), Centro de Biologla Mo- lecular "Severo Ochoa," Universidad Aut6- noma, Canto Blanco, 28049 Madrid, Spain LINDA B. BLOOM (19), Hedco Molecular Biol- ogy Laboratories, Department of Biological Sciences, University of Southern Cali)brnia, Los Angeles, California 90089-1340 ERIK BOYE (45), Department of Biophysics, Institute for Cancer Research, The Norwe- gian Radium Hospital, 0310 Oslo, Norway BONITA J. BREWER (46), Department of Ge- netics, University of Washington, Seattle, Washington 98195-7360 NEAL C. BROWN (4, 17), Department of Phar- macology, University of Massachusetts Medical School Worcester, Massachusetts 01655 GEORGE S. BRUSH (41), Department of Mo- lecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 MARTIN E. BUDD (12), Department of Chem- istry, California Institute of Technology, Pasadena, California 91125 PETER M. J. BURGERS (6), Department o[ Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 6.3110 HONG CAI (2), Hedco Molecular Biology Laboratories, Department of Biological Sci- ences, University of Southern California, Los Angeles, California 90089-1340 CRAIG E. CAMERON (13, 20), Department of Chemistry, The Pennsylvania State Univer- sity, University Park, Pennsylvania 16802 JUDITH L. CAMPBELL (12), Department of. Chemistry and Biology', California Institute o]: Technology, Pasadena, California 91125 X CONTRIBUTORS TO VOLUME 262 TODD L. CAPSON (34), Department of Chemis- try, University of Utah, Salt Lake City, Utah 84132 CHUEN-SHEUE CHIANG (7), Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 GLORIA SHEAU-JIN CHUI (10), Department of Biochemistry, Stanford University, Stan- ford, California 94305-5307 ARTHUR O. CLARK, JR. (15), Center for Ad- vanced Biotechnology and Medicine, and Chemistry Department, Rutgers University, Piscataway, New Jersey 08854-5638 PATRICK CLARK (15), SAIC-Frederick, NCI- Frederick Cancer Research and Develop- ment Center, Frederick, Maryland 21701- 1013 DONALD M. COEN (24), Department of Bio- logical Chemistry and Molecular Pharma- cology, Harvard Medical School, Boston, Massachusetts 02115-5747 FRANK E. J. COENJAERTS (42), Laboratory for Physiological Chemistry, Utrecht Uni- versity, 3508 TA Utrecht, The Netherlands NANCY COLOWICK (44), Department of Mo- lecular Biology, Vanderbilt University, Nashville, Tennessee 37235 WILLIAM C. COPELAND (8, 23), Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324 STEVEN CREIGHTON (19), Hedco Molecular Biology Laboratories, Department of Bio- logical Sciences, University of Southern Cal- ifornia, Los Angeles, California 90089-1340 ELLIOTI" CROOKE (39), Department of Bio- chemistry and Molecular Biology, George- town University Medical Center, Washing- ton, DC 20007 MILLARD G. CULL (3), Department of Bio- chemistry, Biophysics, and Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Den- ver, Colorado 80262 SHIRLEY S. DAUBE (36), Department of Bio- logical Chemistry, The Institute of Life Sci- ences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel ZEGER DEBYSER (35), Department of Biologi- cal Chemistry and Molecular Pharmacol- ogy, Harvard Medical School, Boston, Mas- sachusetts 02115 MELVIN L. DEPAMPHILIS (47), Roche Re- search Center, Roche Institute of Molecular Biology, Nutley, New Jersey 07110 VICTORIA DERBYSHIRE (1, 28), Department of Molecular Biophysics and Biochemistry, Bass Center for Molecular and Structural Biology, Yale University, New Haven, Con- necticut 06520-8114 PAUL DIGARD (24), Department of Pathology, Division of Virology, University of Cam- bridge, Cambridge CB21QP, United Kingdom QUN DONG (8, 23), Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324 KATHLEEN M. DOWNEY (9), Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101 FRITZ ECKSTEIN (16), Max-Planck-Institut flit Experimentelle Medizin, GOttingen, Germany PHILIP J. FAY (21), Departments of Medicine and Biochemistry, University of Rochester, Rochester, New York 14642 TIM FORMOSA (31), Department of Biochem- istry, University of Utah School of Medicine, Salt Lake City, Utah 84132 KATHERINE L. FRIEDMAN (46), Department of Genetics, University of Washington, Seattle, Washington 98195-7360 E. PETER GEiDUSCHEK (37), Department of Biology, University of California, San Diego, La Jolla, California 92093-0634 STEPHEN P. GOFF (27), Department of Bio- chemistry and Molecular Biophysics, How- ard Hughes Medical Institute, Columbia University, College of Physicians and Sur- geons, New York, New York 10032 MYRON F. GOODMAN (2, 19), Hedco Molecu- lar Biology Laboratories, Department of Biological Sciences, University of South- ern California, Los Angeles, California 90089-1340 CONTRIBUTORS TO VOLUME 262 xi DEBORAH M. HINTON (43), Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kid- ney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830 PETER H. VON HIPPEL (36), Institute of Molec- ular Biology, University of Oregon, Eugene, Oregon 97403 LIsa J. HOBBS (43), Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Dis- eases, National Institutes of Health, Bethesda, Maryland 20892-0830 STEPHEN H. HUGHES (15), ABL-Basic Re- search Program, NC1-Frederick Cancer Re- search and Development Center, Frederick, Maryland 21701-1013 ALFREDO JACOBO-MOLINA (15), Center for Advanced Biotechnology and Medicine, and Chemistry Department, Rutgers Uni- versity, Piscataway, New Jersey 08854-5638 THALE C. JARVIS (36), Ribozyme Pharma- ceuticals, Inc., Boulder, Colorado 80308- 7280 CATHERINE M. JOYCE (1, 28), Department of Molecular Biophysics and Biochemistry, Bass Center for Molecular and Structural Biology, Yale University, New Haven, Con- necticut 06520-8114 GEORGE A. KASSAVETIS (37), Department of Biology, University of California, San Diego, La Jolla, California 92093-0634 THOMAS J. KELLY (41), Department of Molec- ular Biology and Genetics, The Johns Hop- kins University School of Medicine, Balti- more, Mar#and 21205 Zw KELMAN (32), Cornell University Medical College, New York, New York 10021 WILLIAM H. KONIGSBER6 (26), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510 THOMAS A. KUNKEL (18), Laboratory of Mo- lecular Genetics, National Institute of Envi- ronmental Health Science, Research Trian- gle Park, North Carolina 27709 JOSE M. LAZARO (5), Centro de Biologia Mo- lecular "Severo Ochoa, " Universidad AutO- noma, Canto Blanco, 28049 Madrid, Spain STUART F. J. LE GRICE (13), Division of In- fectious Diseases, Case Western Reserve University School of Medicine, Cleveland. Ohio 44106-4984 I. R. LEHMAN (7), Department of Biochemis- try, Stanford University School of Medicine, Stanford, California 94305 STUART LINN (10), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 LISA M. MALLABER (21), Departments of Bio- chemistry, Microbiology and Immunology, and the Cancer Center, University of Roch- ester, Rochester, New York 14642 KENNETH J. MARIANS (40), Molecular Biol- ogy Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 STEVEN W. MATSON (29), Department of Biol- ogy, University of North Carolina, Chapel Hill, North Carolina 27599 KEVlN McENTEE (2), Department of Biologi- cal Chemistry and the Molecular Biology Institute, University of California at Los Angeles School of Medicine, Los Angeles', California 90024 CHARLES S. MCHENRY (3), Department of Biochemistry, Biophysics, and Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Den- ver, Colorado 80262 LYNN g. MENDELMAN (30), Department of Biological Chemistry and Molecular Phar- macology, Harvard University Medical School, Boston, Massachusetts 02115 PAUL G. MITSIS (7), Department of Biochem- istry, Stanford University School of Medi- cine, Stanford, California 94305 ROBB E. MosEs (38), Department of Molecu- lar and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201 GISELA MOSIC (44), Department of Molecular Biology, Vanderbilt University, Nashville. Tennessee 37235 GRE6ORY P. MULLEN (14), Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 xii CONTRIBUTORS TO VOLUME 262 VYTAUTAS NAKTINIS (32), Institute of Bio- technology, V. Graiciuno 8, 2028 Vilnius, Lithuania NANCY G. NOSSAL (34, 43), Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kid- ney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830 MIKE O'DONNELL (32, 33), Howard Hughes Medical Institute, CorneU University Medi- cal College, New York, New York 10021 JULIA K. PINSONNEAULT (28), Department of Molecular Biophysics and Biochemistry, Bass Center for Molecular and Structural Biology, Yale University, New Haven, Con- necticut 06520-8114 MICHAEL K. REDDY (36), Department of Chemistry, University of Wisconsin- Milwaukee, Milwaukee, Wisconsin 53201- 0413 LINDA J. REHA-KRANTZ (25), Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9 Canada EARS ROGGE (8), Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324 MARGARITA SALAS (5, 22), Cenlro de Bio- logla Molecular "Severo Ochoa," Universi- dad Aut6noma, Canto Blanco, 28049 Ma- drid, Spain KIRSTEN SKARSTAD (45), Department of Bio- physics, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway ANTERO G. So (9), Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101 PETER SPACCIAPOLI (43), Laboratory of Mo- lecular and Cellular Biology, National Insti- tute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830 BRUCE STILLMAN (41), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 ALICE TELESNITSKY (27), Department of Mi- crobiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620 JAMES B. THOMSON (16), Max-Planck-lnstitut fiir Experimentelle Medizin, GOttingen, Germany RACHEL L. TINKER (37), Department of Biol- ogy, University of California, San Diego, La Jolla, California 92093-0634 JENNIFER TURNER (33), Cornell University Medical College, New York, New York 10021 PETER C. VAN DER VLIET (42), Laboratory for Physiological Chemistry, Utrecht Uni- versity, 3508 TA Utrecht, The Netherlands TERESA S F. WANG (8, 23), Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324 STEPHEN E. WEITZEE (36), Institute of Molec- ular Biology, University of Oregon, Eugene, Oregon 97403 SAMUEL H. WILSON (11 ), Sealy Center for Mo- lecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068 JACQUEEINE WITTMEYER (31), Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132 GEORGE E. WRIGHT (17), Department of Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 HONG YU (2), Hedco Molecular Biology Lab- oratories, Department of Biological Sci- ences, University of Southern California, Los Angeles, California 90089-1340 [1] DNA POLYMERASE I AND KLENOW FRAGMENT 3 [ 1] Purification of Escherichia coli DNA Polymerase I and Klenow Fragment By CATHERINE M. JOYCE and VICTORIA DERBYSHIRE Introduction DNA polymerase I (Pol I) of Escherichia coli, the first DNA polymerase to be discovered, has long served as a simple model system for studying the enzymology of DNA synthesis. ~ The original studies of Pol I relied on purification of the enzyme from E. coli extracts without genetic manipula- tion, yielding around 10 mg of purified enzyme per kilogram of cell paste. 2 Cloning ofpolA, the structural gene for Pol I, in a variety of phage A vectors increased the level of expression about 100-fold. 3"4 Sequence analysis of the cloned polA gene 5 allowed construction of a plasmid-derived expression system for the Klenow fragment portion of Pol I, 6 comprising the C-terminal two-thirds of the protein and having the polymerase and 3' ~ 5' (proofread- ing)-exonuclease functions of the parent molecule, but lacking the 5' Y-exonuclease that is used in nick-translation. (Earlier attempts to express whole Pol I on a plasmid vector were unsuccessful because of the lethality of wild-type polA in multiple copies, 3 and indicated the need for more sophisticated vectors giving tight control of the level of expression.) The ability to purify large quantities of Klenow fragment paved the way for the determination of its structure by X-ray crystallography] In addition to their importance as experimental systems in their own right, both Pol I and Klenow fragment have found extensive use as biochemical reagents in a variety of cloning, sequencing, and labeling procedures. Over the years we have made improvements in the expression systems for Pol I and Klenow fragment; we describe here our most recent constructs and protocols, which typically give yields of 10 mg of pure polymerase per gram of cells. ~A. Kornberg and T. A. Baker, "DNA Replication," p. 113. Freeman, San Francisco (1992). T. M. Jovin, P. T. Englund, and L. L. Bertsch, J. Biol. Chem. 244, 2996 (1969). W. S. Kelley, K. Chalmers, and N. E. Murray, Proc. Natl. Acad. Sci. USA 74, 5632 (1977). 4 N. E. Murray and W. S. Kelley, Molec. Gen. Genet. 175, 77 (1979). 5 C. M. Joyce, W. S. Kelley, and N. D. F. Grindley, J. Biol. Chem, 257, 1958 (1982). C. M. Joyce and N. D. F. Grindley, Proc. Natl. Acad. Sci. USA 80, 1830 (1983). 7 D. L. Ollis, P. Brick, R. Hamlin, N. G. Xuong, and T. A. Steitz, Nature 313, 762 (1985). Copyright © 1995 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 262 All rights of reproduction in any form reserved. 4 DNA POLYMERASES [ II Expression Plasmids Both whole Pol I and Klenow fragment have been substantially overex- pressed using constructs derived from the pAS1 vector, 8 in which transcrip- tion is driven from the strong leftward promoter (PO of phage A, and the translational start signals are derived from the AcII gene. For expression of Klenow fragment, the ATG initiation codon of the expression vector replaces the codon for Val-324(GTG), the N-terminal amino acid of Klenow fragment. The construction of this plasmid has already been described? It gives about a tenfold higher expression of Klenow fragment than the origi- nal expression plasmid in which the translational signals were less well optimized. 6 In the Pol I expression plasmid, whose construction is described elsewhere, the vector-derived ATG codon replaces the natural GTG start of the polA gene and no upstream polA DNA is present. This plasmid gives a much higher level of expression than the Pol I expression plasmid described previously by Minkley et al. 1° Not only did the earlier plasmid use the rather poor poIA translational initiation signals, but it also retained DNA sequences derived from the poIA promoter. Because of the lethality of a nonrepressed polA gene at high copy number, the latter sequences are probably responsible for the considerable problems of plasmid instability reported by Minkley eta/. 1° Host Strains The highest levels of expression that we have achieved were in a strain background such as ARI20, u in which expression is controlled by the wild- type h repressor on a defective prophage. SOS-induction using nalidixic acid results in recA-mediated cleavage and inactivation of the repressor, leading to expression of the PL-driven target gene. However, this system is not appropriate for expressing mutant derivatives of Po]I or Klenow fragment. Because the expression vector requires a wild-type chromosomal copy of poIA for its replication, it is desirable, when expressing a mutant protein, to use a recA-defective host in order to minimize the possibility that exchange between plasmid and chromosomal poIA sequences might eliminate the mutant information. Because nalidixic acid induction is ruled out in a recA- background, we use heat induction of a strain carrying the 8 M. Rosenberg, Y S. Ho, and A. Shatzman, Meth. Enzymol. 101, 123 (1983). 9 A. H. Polesky, T. A. Steitz, N. D. F. Grindley, and C. M. Joyce, J. Biol. Chem. 265, 14579 (1990). 10 E. G. Minkley, Jr., A. T. Leney, J. B. Bodner, M. M. Panicker, and W. E. Brown, J. BioL Chem. 259, 10386 (1984). 11 j. E. Mott, R. A. Grant, Y S. Ho, and T. Platt, Proc. Natl. Acad. Sei. USA 82, 88 (1985). [1] DNA POLYMERASE ! AND KLENOW FRAGMENT 5 TABLE I OVERPRODUCER STRAINS FOR DNA POLYMERASE I AND KLENOW FRAGMENT Protein Plasmid Host Strain number Inducing treatment Pol I pCJ194 AR120 CJ402 Nalidixic acid Pol I" pCJ194" CJ376 Heat Klenow fragment pCJ122 AR120 CJ333 Nalidixic acid Klenow fragment pCJ122 CJ378 CJ379 Heat Klenow fragment" pCJ122" CJ376 Heat "Or mutant derivatives. clss7 temperature-sensitive A repressor. Our host strain, CJ376, 9 is recA and carries the ci857 allele on a chloramphenicol-resistant plasmid, pCJ136, which is compatible with the expression vector. The CJ376 host strain is also deficient in exonuclease III, which has in the past caused concern as a possible contaminant in the purification, 12 but is now largely irrelevant with the high-resolution chromatographic methods described here. Note that the availability of the ci857 gene on a compatible plasmid means that virtually any strain can be converted into an expression host merely by transformation; for example, the host CJ378, obtained by transformation of BW9109,13 is recA + and deficient in exonuclease III, and provides a good background for heat induction of wild-type Klenow fragment. Induction Protocols Typical procedures follow for the growth and induction of 1 to 2 liters of cells. The procedure can easily be scaled up, for example, for use in a fermentor. Although we routinely maintain selection pressure for the Amp R determinant as a precaution against loss of the expression plasmid, we have not found plasmid instability to be a serious problem in this system. Strains The overproducer strains currently in use are listed in Table I. They are stored as glycerol cultures at 20°. 14 Before use they should be streaked out on plates containing carbenicillin (50/,~g/ml) and, when using the CJ376 or CJ378 host, chloramphenicol (15/zg/ml). The incubation temperature is 30 ° for the heat-inducible strains, and 37 ° for the others. Strains containing 12 p. Setlow, Methods Enzymol. 29, 3 (1974). 13 B. J. White, S. J. Hochhauser, N. M. Cintr6n, and B. Weiss, J. Bacteriol. 126, 1082 (1976). 14 j. H. Miller, "Experiments in Molecular Genetics." Cold Spring Harbor Laboratories, Cold Spring Harbor (1972). 6 DNA POLYMERASES [ 11 overproducer plasmids for mutant polymerase derivatives are not stored as such; to minimize the chances for exchange between wild-type and mutant information, the mutated overproducer plasmid is introduced into the CJ376 (recA) host only when needed. Media LB: 10 g tryptone, 5 g yeast extract, and 5 g NaC1 per liter. 14 MIM (maximal induction medium)11:32 g tryptone and 20 g yeast extract, adjusted to pH 7.6 with 3 M NaOH, in a total volume of 900 ml. After autoclaving, 100 ml 10 x M9 salts, 0.1 ml 1 M MgSO4, and 0.1 ml 0.01 M FeC13 are added. 10 x M9 salts14:6 g Na2HPO4, 3 g KH2PO4, 5 g NaCI, and 10 g NH4C1 dissolved in H20 to a total volume of 100 ml, and autoclaved. Nalidixic acid: 0.1 g nalidixic acid in 10 ml 0.3 M NaOH, filter-sterilized and stored at 4 ° . Carbenicillin: 50 mg/ml in H20, filter-sterilized and stored at 4 °. All media are supplemented with carbenicillin at 50 ~g/ml. Ampicillin, or other related antibiotics, can be substituted. Nalidixic Acid Induction A 1-ml inoculum is grown from a single colony of the appropriate overproducer strain in LB/carbenicillin at 37 ° for approximately 8 hr. This is diluted into 40 ml MIM/carbenicillin and grown overnight. Half of this culture is inoculated into each of two 2-liter baffle flasks containing 500 ml MIM/carbenicillin. These are grown at 37 ° with vigorous aeration (about 250 rpm in a New Brunswick series 25 incubator shaker) to OD600 ~ 1. Nalidixic acid (2 ml per 500 ml culture) is added, giving a final concentration of 40/zg/ml. The cells (typically 5 to 6 g) are harvested by centrifugation about 8 hr later, washed with cold 50 mM Tris-HC1, pH 7.5, and stored frozen at -70 ° . Heat Induction A 1-ml inoculum is grown from a single colony of the appropriate overproducer strain in LB/carbenicillin at 30 ° for approximately 8 hr, and then diluted into 50 ml of the same medium and grown overnight. Half of this culture is inoculated into each of two 2-liter baffle flasks containing 750 ml of LB/carbeniciUin. These are grown at 30 ° with vigorous aeration to an OD60o ~ 0.6 (approximately 4 hr). The temperature is raised by the addition to each flask of 250 ml LB, previously heated to 90 °, and the flask is transferred to a shaker at 42 °. After a further 2 hr, the cells (typically 3 to 5 g) are harvested as described earlier. [1] DNA POLYMERASE I AND KLENOW FRAGMENT 7 Monitoring Induction For either induction method a 1-ml sample of the culture should be taken just before the inducing treatment, and when the cells are harvested. The sample is spun for 2 rain in a microfuge, and the pelleted cells are resuspended in 50/zl of SDS-PAGE sample buffer and lysed by heating for 2 to 3 rain at 100 °. A 5- to 10-/xl sample of this whole cell lysate is examined by SDS-PAGE, using a 10% gel for Klenow fragment and an 8% gel for whole Pol I. Typical results are shown in Fig. 1. Purification Method for Klenow Fragment or DNA Polymerase I The two methods are identical, except where noted. The procedure described here makes use of the Pharmacia fast protein liquid chromatogra- phy (FPLC) system. If this equipment is not available, published proce- dures 6'1° using conventional chromatography are also satisfactory. Klenow fragment Pol I Nal Heat Nal Heat t= 0 7.5 0 1 2 hours iiii i!!iiiiii :i~i{ii ¸ iii~!~iii i!ii! FIG. 1. Overproduction of Klenow fragment and DNA polymerase I. The Klenow fragment panel shows SDS-PAGE analysis of whole cell extracts of appropriate overproducer strains, sampled before induction (t = 0) and at the indicated times after the inducing treatment. The Pol I panel shows samples of the clarified crude cell lysates from cells expressing whole Pol I, after induction with nalidixic acid or with heat. The arrows indicate the positions of the respective expressed products. [...]... an associated 3' -~ 5'-exonuclease activity that can be assayed by measuring hydrolysis of single-stranded DNA: Single-stranded DNA, ~ DNA, , ~ + dNMP Pol II (0.1 to 1 unit) is added to 40 txl 5'-32p-labeled single-stranded DNA reaction solution [180 nM 5'-32p-labeled single-stranded synthetic DNA oligonucleotide having an arbitrary uniform length, approximately 5 ~Ci/ pmol, 7.3 mM MgC12, 1 mM DTT,... (1994) [2] E coli DNA eoL n 15 Procedure Deoxyribonucleotide Incorporation A s s a y DNA polymerase activity is assayed by measuring the incorporation of [3H]dTMP into acid-insoluble DNA The reaction mixture (0.05 ml) contains 2.5 mM dithiothreitol (DTT), 20 mM Tris-HC1 (pH 7.5), 7.3 mM MgC12, 6 mM spermidine hydrochloride, 1 mg/ml bovine serum albumin (BSA), 1.1 mM gapped primer-template DNA, 60/xM dATP,... 20 n M 9'22 Even with substrates that permit extensive DNA synthesis, both Pol I and Klenow fragment have rather low processivity, adding in the range of 10 to 50 nucleotides for each enzyme -DNA encounter.9,21,23-25 The 3' ~ 5'-exonuclease can be assayed on a variety of single-stranded or double-stranded DNA substrates 12'17On single-stranded DNA, the specific activity of the 3' ~ 5'-exonuclease of... dimer, permitting it to slide rapidly down the DNA that it presumably encircles but preventing it from readily dissociating Protein-protein contacts between/3 and other components of the replicative complex tether the polymerase to the DNA, increasing its processivity (3) A five-protein DnaX complex recognizes primer termini and closes the/3 bracelet around DNA This complex remains firmly associated as... GF/C filters (MiUipore, Cat No 1822 024) M13Gori DNA, single-stranded binding protein (SSB), and dnaG primase (listed below) are obtained from ENZYCO, Inc., Denver, CO Each 25-tzl reaction contains, in order of addition: Assay component Enzyme dilution buffer (EDB) 250 mM Magnesium acetate M13Gori DNA (0D260 = 2) SSB dNTP cocktail rNTP cocktail Rifampicin DnaG primase tzl 14 1 Final concentration Approximately... the product DNA 22 At a more subtle level, the immediate DNA sequence context surrounding the primer terminus also exerts an influence on the kinetic parameters, so that there is variation (within a fairly narrow range) in the values obtained using different experimental systems Typically, the Km for dNTP utilization is in the range of 1 to 5 /zM, 9'22'23 and the dissociation constant for DNA binding... albumin (BSA), 1.1 mM gapped primer-template DNA, 60/xM dATP, dCTP, dGTP, [3H]dTTP (5 × 107 t o 1 × 108 cpm/txmol), and 0.5 to 5 units of enzyme Gapped primer-template DNA refers to salmon sperm DNA digested to about 15% acid solubility with DNase I 15 Reactions are incubated for 15 rain at 37° and are terminated by the addition of cold 0.2 M sodium pyrophosphate in 15% trichloroacetic acid One Pol II polymerase... chromosomal replication and control of spontaneous mutagenesis It is comprised of a 10 subunit holoenzyme complex, including the c~ subunit containing 5' ~ 3'-polymerase activity, the e subunit containing 3' ~ 5' (proofreading)-exonuclease activity, and a multisubunit y complex and/~ protein required for enzyme processivity, z E coli Pol II was discovered in 1970, 3 yet its role in DNA replication. .. single-stranded DNA ~'~ One Pol II exonuclease unit catalyzes the reduction of 1 pmol/min of single~5A E Oleson and J F Koerner,J Biol Chem 239, 2935 (1964) 16M S Boosalis,J Petruska, and M F, Goodman,J Biol Chem 262, 14,689 (1987) 17H Cai, L B Bloom,R Eritja, and M F Goodman,J Biol Chem 268, 23,567 (1993) ISN Muzyczka,R L Poland, and M J Bessman.J Biol Chem 247, 7116 (1972) 16 DNA POLYMERASES [2] stranded DNA. .. dNMP into acid-soluble material at 37° A "turnover" assay can be used to measure the action of the Y-exonuclease coupled to DNA synthesis This assay measures the DNA- dependent conversion of dNTP to its corresponding dNMP, as described previously.18 Purification of Escherichia coli DNA Polymerase II Cell Growth E coli JM109 cells carrying the Pol II (polB) gene on an overproducing plasmid pHY400 (wild-type . of faithful copying of the genome by the self-editing DNA replication and repair apparatus. Past study of the enzymes involved in DNA replication has given rise to a number of highly refined. transcription, and begin- ning to study the coupling of these processes to DNA replication. Methods of analyzing DNA replication in vivo are also included. JUDITH L. CAMPBELL xiii Contributors. precursors into high molecular weight DNA. 12 Either "activated" calf thymus DNA (made by nicking with DNase I) or poly [d(AT)] can be used as the DNA substrate; poly[d(AT)], being available