DNA Replication

731 Pages·1995·19.607 MB·English

by  Campbell J. (ed.), Abelson J. (ed.), Simon M.I. (ed.)

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
elcitrA srebmun era ni sesehtnerap gniwollof the seman of .srotubirtnoc
snoitailiffA detsil era .tnerruc
EDWARD ARNOLD (15), Center for Advanced Hughes Medical Institute, Columbia Uni-
Biotechnology and Medicine, and Chemis- versity, College of Physicians and ,'snoegruS
try Department, Rutgers University, Piscata- New York, New York 23.001
way, New Jersey 8365-45880 Luxs BLANCO (5, 22), Centro de Biologla Mo-
ROBERT A. BAMBARA (21), Departments of lecular "Severo Ochoa," Universidad Aut6-
Biochemistry, Microbiology and Immunol- noma, Canto Blanco, 28049 Madrid, Spain
ogy, and the Cancer Center, University of LINDA B. BLOOM (19), Hedco Molecular Biol-
Rochester, Rochester, New York 24641 ogy Laboratories, Department of Biological
MARJORIE H. BARNES (4), Department of Sciences, University of Southern ,ainrb)ilaC
Pharmacology, University of Massachusetts Los Angeles, California 0431-98009
Medical School, Worcester, Massachusetts ERIK BOYE (45), Department of Biophysics,
55610 Institute for Cancer Research, The Norwe-
BLAINE WEMOLOHTRAB (37), Department of gian Radium Hospital, 0310 Oslo, Norway
Medical Biochemistry, Southern Illinois ATINOB J. BREWER (46), Department of Ge-
University School of Medicine, Carbondale, netics, University of Washington, Seattle,
Illinois 3.056-10926 Washington 0637-59189
DANIEL W. BEAN (29), Department of Biol- NEAL C. NWORB (4, 17), Department of Phar-
ogy, University of North Carolina, Chapel macology, University of Massachusetts
Hill, North Carolina 27599 Medical School Worcester, Massachusetts
WILLIAM A. BEARD (11), Scaly Center for 55610
Molecular Science, University of Texas GEORGE S. BRUSH (41), Department of Mo-
Medical Branch, Galveston, Texas -55577 lecular Biology and Genetics, The Johns
8601 Hopkins University School of Medicine,
KATARZyNA BEBENEK (18), Laboratory of Baltimore, Maryland 50212
Molecular Genetics, National Institute of MARTIN E. BUDD (12), Department of Chem-
Environmental Health Science, Research istry, California Institute of Technology,
Triangle Park, North Carolina 27709 Pasadena, California 52119
MAILLIW R. BEBRIN (24), Department of Bio- PETER M. J. BURGERS (6), Department o
logical Chemistry and Molecular Pharma- Biochemistry and Molecular Biophysics,
cology, Harvard Medical School, Boston, Washington University School of Medicine,
Massachusetts 7475-51120 .tS Louis, Missouri 0113.6
NEHPETS J. BENKOVlC (13, 20, 34), Depart- HONG CAI (2), Hedco Molecular Biology
ment of Chemistry, The Pennsylvania State Laboratories, Department of Biological Sci-
University, University Park, Pennsylvania ences, University of Southern California,
20861 Los Angeles, California 0431-98009
ROLF BERNANDER (45), Department of Bio- CRAIG E. CAMERON (13, 20), Department of
physics, Institute for Cancer Research, The Chemistry, The Pennsylvania State Univer-
Norwegian Radium Hospital 0310 Oslo, sity, University Park, Pennsylvania 20861
Norway JUDITH L. CAMPBELL (12), Department .fo
YCATS BLAIN (27), Department of Biochemis- Chemistry and Biology', California Institute
try and Molecular Biophysics, Howard :o Technology, Pasadena, California 52119
ix
X CONTRIBUTORS TO VOLUME 262
DDOT L. NOSPAC (34), Department of Chemis- REGEZ RESYBED (35), Department of Biologi-
try, University of Utah, Salt Lake City, cal Chemistry and Molecular Pharmacol-
Utah 23148 ogy, Harvard Medical School, Boston, Mas-
sachusetts 51120
EUEHS-NEUHC CHIANG (7), Department of
Biochemistry, Stanford University School NIVLEM L. SILIHPMAPED (47), Roche Re-
of Medicine, Stanford, California 50349 search Center, Roche Institute of Molecular
Biology, Nutley, New Jersey 01170
AIROLG NIJ-UAEHS CHUI (10), Department of
Biochemistry, Stanford University, Stan- AIROTCIV ERIHSYBRED (1, 28), Department
ford, California 7035-50349 of Molecular Biophysics and Biochemistry,
Bass Center for Molecular and Structural
RUHTRA O. ,KRALC JR. (15), Center for Ad-
Biology, Yale University, New Haven, Con-
vanced Biotechnology and Medicine, and
Chemistry Department, Rutgers University, necticut 4118-02560
Piscataway, New Jersey 8365-45880 LUAP DIGARD (24), Department of Pathology,
KCIRTAP CLARK (15), SAIC-Frederick, NCI- Division of Virology, University of Cam-
Frederick Cancer Research and Develop- bridge, Cambridge CB21QP, United
ment Center, Frederick, Maryland 21701- Kingdom
3101 NUQ DONG (8, 23), Department of Pathology,
DLANOD M. COEN (24), Department of Bio- Stanford University School of Medicine,
logical Chemistry and Molecular Pharma- Stanford, California 4235-50349
cology, Harvard Medical School, Boston, NEELHTAK M. YENWOD (9), Department of
Massachusetts 7475-51120 Medicine, University of Miami School of
Medicine, Miami, Florida 10133
KNARF E. J. STREAJNEOC (42), Laboratory
for Physiological Chemistry, Utrecht Uni- FRITZ NIETSKCE (16), Max-Planck-Institut
versity, 3508 TA Utrecht, The Netherlands flit Experimentelle Medizin, GOttingen,
Germany
YCNAN KCIWOLOC (44), Department of Mo-
lecular Biology, Vanderbilt University, PILIHP J. YAF (21), Departments of Medicine
Nashville, Tennessee 37235 and Biochemistry, University of Rochester,
Rochester, New York 24641
MAILLIW C. DNALEPOC (8, 23), Department
of Pathology, Stanford University School of MIT ASOMROF (31), Department of Biochem-
Medicine, Stanford, California 4235-50349 istry, University of Utah School of Medicine,
Salt Lake City, Utah 23148
NEVETS NOTHGIERC (19), Hedco Molecular
Biology Laboratories, Department of Bio- ENIREHTAK L. NAMDEIRF (46), Department of
logical Sciences, University of Southern Cal- Genetics, University of Washington, Seattle,
ifornia, Los Angeles, California 0431-98009 Washington 0637-59189
"ITOILLE EKOORC (39), Department of Bio- E. PETER KEHCSUDiEG (37), Department of
chemistry and Molecular Biology, George- Biology, University of California, San
town University Medical Center, Washing- Diego, La Jolla, California 4360-39029
ton, DC 20007 NEHPETS P. GOFF (27), Department of Bio-
DRALLIM G. CULL (3), Department of Bio- chemistry and Molecular Biophysics, How-
chemistry, Biophysics, and Genetics and ard Hughes Medical Institute, Columbia
Program in Molecular Biology, University University, College of Physicians and Sur-
of Colorado Health Sciences Center, Den- geons, New York, New York 23001
ver, Colorado 26208 NORYM F. NAMDOOG (2, 19), Hedco Molecu-
YELRIHS S. DAUBE (36), Department of Bio- lar Biology Laboratories, Department of
logical Chemistry, The Institute of Life Sci- Biological Sciences, University of South-
ences, The Hebrew University of Jerusalem, ern California, Los Angeles, California
Givat-Ram, Jerusalem ,40919 Israel 0431-98009
CONTRIBUTORS TO VOLUME 262 xi
DEBORAH M. HINTON (43), Laboratory of STUART F. J. LE GRICE (13), Division of -nI
Molecular and Cellular Biology, National fectious Diseases, Case Western Reserve
Institute of setebaiD and Digestive and Kid- ytisrevinU School of ,enicideM .dnalevelC
ney ,sesaesiD National Institutes of ,htlaeH Ohio 4894-60144
,adsehteB Maryland 0380-29802 I. R. LEHMAN (7), Department of Biochemis-
PETER H. NOV HIPPEL (36), Institute of -celoM ,yrt Stanford ytisrevinU School of ,enicideM
ralu Biology, ytisrevinU of ,nogerO ,eneguE Stanford, California 50349
nogerO 30479 STUART LINN (10), Department of raluceloM
LIsa J. HOBBS (43), Laboratory of raluceloM and Cell Biology, University of ,ainrofilaC
and Cellular Biology, National Institute of Berkeley, California 02749
Diabetes and Digestive and Kidney Dis-
LISA M. MALLABER (21), Departments of -oiB
eases, National Institutes of Health,
,yrtsimehc Microbiology and Immunology,
,adsehteB Maryland 0380-29802
and eht Cancer ,retneC ytisrevinU of Roch-
STEPHEN H. HUGHES (15), ABL-Basic -eR ,retse Rochester, New York 24641
hcraes Program, kcirederF-1CN recnaC Re-
KENNETH J. MARIANS (40), Molecular Biol-
hcraes dna Development ,retneC ,kcirederF
ogy Program, Memorial Sloan-Kettering
Maryland 3101-10712
recnaC ,retneC New York, New York 12001
ALFREDO JACOBO-MOLINA (15), Center for
NEVETS W. MATSON (29), Department of -loiB
Advanced Biotechnology and ,enicideM
ogy, University of North ,aniloraC Chapel
and Chemistry Department, Rutgers Uni-
,lliH North Carolina 99572
,ytisrev ,yawatacsiP New Jersey 8365-45880
KEVlN McENTEE (2), Department of -igoloiB
THALE C. JARVIS (36), Ribozyme Pharma-
lac Chemistry and eht Molecular Biology
,slacituec Inc., Boulder, Colorado -80308
Institute, University of California at Los
0827
Angeles School of ,enicideM Los ,'selegnA
CATHERINE M. JOYCE (1, 28), Department of
ainrofilaC 42009
Molecular Biophysics and Biochemistry,
Bass Center for Molecular and larutcurtS CHARLES S. MCHENRY (3), Department of
Biology, Yale ,ytisrevinU New Haven, Con- ,yrtsimehcoiB Biophysics, and sciteneG and
necticut 4118-02560 Program ni Molecular Biology, ytisrevinU
of Colorado Health Sciences ,retneC Den-
GEORGE A. KASSAVETIS (37), Department of
,rev Colorado 26208
Biology, University of California, naS
,ogeiD La Jolla, California 4360-39029 LYNN g. MENDELMAN (30), Department of
Biological Chemistry and Molecular Phar-
THOMAS J. KELLY (41), Department of -celoM
macology, Harvard University Medical
ular Biology and ,sciteneG ehT Johns Hop-
School, Boston, sttesuhcassaM 51120
kins University School of Medicine, Balti-
more, Mar#and 50212 PAUL G. MITSIS (7), Department of Biochem-
Zw KELMAN (32), Cornell ytisrevinU lacideM ,yrtsi Stanford University School of -ideM
,egelloC New York, New York 12001 ,enic Stanford, California 50349
WILLIAM H. KONIGSBER6 (26), Department ROBB E. MosEs (38), Department of -uceloM
of Molecular Biophysics and ,yrtsimehcoiB ral and Medical Genetics, Oregon Health
Yale ,ytisrevinU New Haven, Connecticut Sciences University, Portland, Oregon
01560 10279
THOMAS A. KUNKEL (18), Laboratory of Mo- GISELA MOSIC (44), Department of raluceloM
ralucel ,sciteneG National Institute of Envi- Biology, Vanderbilt ,ytisrevinU .ellivhsaN
ronmental Health ,ecneicS Research -nairT eessenneT 53273
elg Park, North Carolina 90772 GRE6ORY P. MULLEN (14), Department of
JOSE M. LAZARO (5), Centro ed Biologia Mo- Biochemistry, University of Connecticut
ralucel "Severo ,aohcO " dadisrevinU -OtuA Health Center, Farmington, Connecticut
noma, Canto Blanco, 28049 ,dirdaM Spain 23060
xii CONTRIBUTORS TO VOLUME 262
SATUATYV SINITKAN (32), Institute of Bio- Diseases, National Institutes of Health,
technology, .V Graiciuno ,8 2028 Vilnius, Bethesda, Maryland 0380-29802
Lithuania ECURB NAMLLITS (41), Cold Spring Harbor
YCNAN G. LASSON (34, 43), Laboratory of Laboratory, Cold Spring Harbor, New
Molecular and Cellular Biology, National York 42711
Institute of Diabetes and Digestive and Kid- ECILA YKSTINSELET (27), Department of Mi-
ney Diseases, National Institutes of Health, crobiology and Immunology, University of
Bethesda, Maryland 0380-29802 Michigan Medical School, Ann Arbor,
EKIM LLENNOD'O (32, 33), Howard Hughes Michigan 0260-90184
Medical Institute, CorneU University Medi- SEMAJ B. NOSMOHT (16), Max-Planck-lnstitut
cal College, New York, New York 12001 fiir Experimentelle Medizin, GOttingen,
Germany
AILUJ K. TLUAENNOSNIP (28), Department of
Molecular Biophysics and Biochemistry, LEHCAR L. REKNIT (37), Department of Biol-
Bass Center for Molecular and Structural ogy, University of California, San Diego,
Biology, Yale University, New Haven, Con- La Jolla, California 4360-39029
necticut 4118-02560 REFINNEJ RENRUT (33), Cornell University
Medical College, New York, New York
LEAHCIM K. REDDY (36), Department of
12001
Chemistry, University of Wisconsin-
Milwaukee, Milwaukee, Wisconsin 53201- RETEP C. NAV RED VLIET (42), Laboratory
3140 for Physiological Chemistry, Utrecht Uni-
versity, 3508 TA Utrecht, The Netherlands
ADNIL J. ZTNARK-AHER (25), Department of
Biological Sciences, University of Alberta, ASERET S.-F. WANG (8, 23), Department of
Edmonton, Alberta T6G 2E9 Canada Pathology, Stanford University School of
Medicine, Stanford, California 4235-50349
SRAE ROGGE (8), Department of Pathology,
NEHPETS E. EEZTIEW (36), Institute of Molec-
Stanford University School of Medicine,
ular Biology, University of Oregon, Eugene,
Stanford, California 4235-50349
Oregon 30479
ATIRAGRAM SALAS (5, 22), Cenlro de Bio-
LEUMAS H. NOSLIW (11 ), Sealy Center for Mo-
logla Molecular "Severo Ochoa," Universi-
lecular Science, University of Texas Medical
dad Aut6noma, Canto Blanco, 28049 Ma-
Branch, Galveston, Texas 8601-55577
drid, Spain
ENIEEUQCAJ REYEMTTIW (31), Department of
NETSRIK DATSRAKS (45), Department of Bio- Biochemistry, University of Utah School of
physics, Institute for Cancer Research, The Medicine, Salt Lake City, Utah 23148
Norwegian Radium Hospital, 0310 Oslo,
EGROEG E. WRIGHT (17), Department of
Norway
Pharmacology, University of Massachusetts
ORETNA G. So (9), Department of Medicine, Medical School, Worcester, Massachusetts
University of Miami School of Medicine, 55610
Miami, Florida 10133
GNOH YU (2), Hedco Molecular Biology Lab-
RETEP ILOPAICCAPS (43), Laboratory of Mo- oratories, Department of Biological Sci-
lecular and Cellular Biology, National Insti- ences, University of Southern California,
tute of Diabetes and Digestive and Kidney Los Angeles, California 0431-98009
1
DNA POLYMERASE I AND KLENOW FRAGMENT 3
1 Purification of Escherichia coli DNA Polymerase I and
Klenow Fragment
By CATHERINE M. JOYCE and AIROTCIV DERBYSHIRE
Introduction
DNA polymerase I (Pol I) of aihcirehcsE ,iloc 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. 4"3 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).
thgirypoC © 5991 yb cimedacA Press, .cnI
SDOHTEM NI ,YGOLOMYZNE .LOV 262 llA rights of noitcudorper ni yna form .devreser
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
ehT tsehgih slevel of noisserpxe that ew have deveihca were ni a niarts
dnuorgkcab hcus sa ARI20, u ni hcihw noisserpxe si dellortnoc by eht -dliw
epyt h rosserper no a evitcefed .egahporp noitcudni-SOS using cixidilan
dica results ni detaidem-Acer egavaelc dna noitavitcani of eht ,rosserper
gnidael to noisserpxe of the nevird-LP target gene. ,revewoH this metsys
si not etairporppa for gnisserpxe mutant sevitavired of PoI ro wonelK
.tnemgarf esuaceB eht noisserpxe rotcev seriuqer a epyt-dliw lamosomorhc
ypoc of AIop for sti ,noitacilper it si ,elbarised when gnisserpxe a mutant
,nietorp to esu a evitcefed-Acer host ni order to eziminim eht ytilibissop
that egnahcxe between dimsalp dna lamosomorhc poIA secneuqes thgim
etanimile the mutant .noitamrofni esuaceB cixidilan acid noitcudni si ruled
out ni a -Acer ,dnuorgkcab ew esu heat induction of a strain gniyrrac eht
8 .M ,grebnesoR .S-.Y Ho, dna .A ,namztahS .hteM Enzymol. ,101 321 .)3891(
9 .A H. Polesky, .T A. Steitz, .N .D .F Grindley, and .C .M Joyce, .J Biol. Chem. ,562
97541 .)0991(
01 E. .G Minkley, Jr., A. .T Leney, .J .B Bodner, .M .M Panicker, dna .W .E ,nworB .J LoiB
.mehC ,952 68301 .)4891(
11 j. E. Mott, R. A. Grant, .S-.Y Ho, and .T Platt, .corP .ltaN Acad. Sei. ASU ,28 88 .)5891(
1 DNA POLYMERASE ! AND KLENOW FRAGMENT 5
TABLE I
RECUDORPREVO SNIARTS ROF DNA ESAREMYLOP I DNA WONELK TNEMGARF
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 si
also deficient in exonuclease III, which has in the past caused concern as
a possible contaminant in the purification, 21 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 31,9019WB 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°. 41 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 73 ° for the others. Strains containing
21 p. Setlow, Methods Enzymol. 29, 3 (1974).
31 B. J. White, .S J. Hochhauser, N. M. Cintr6n, and B. Weiss, J. Bacteriol. 126, 1082 (1976).
41 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: 01 g tryptone, 5 g yeast extract, and 5 g NaC1 per liter. 41
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
009 ml. After autoclaving, 001 ml 01 x M9 salts, 1.0 ml 1 M MgSO4,
and 1.0 ml 0.01 M FeC13 are added.
01 x M9 salts14:6 g Na2HPO4, 3 g KH2PO4, 5 g NaCI, and 01 g 1C4HN
dissolved in H20 to a total volume of 001 ml, and autoclaved.
Nalidixic acid: 1.0 g nalidixic acid in 01 ml 3.0 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 05 ~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 73 ° 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 73 ° with vigorous aeration (about
250 rpm in a New Brunswick series 52 incubator shaker) to 006DO ~ .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 05 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 o06DO ~ 0.6 (approximately 4 hr). The temperature is raised by the
addition to each flask of 250 ml LB, previously heated to 09 ,° 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
gnirotinoM 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 °1'6 using conventional chromatography are also satisfactory.
Klenow fragment Pol I
Nal Heat Nal Heat
t= 0 7.5 0 1 2 hours
iiii iiiiii!!i
!ii!i
ii{i~i: ii¸ i~!~iii
.GIF .1 Overproduction of and fragment Klenow DNA fragment Klenow The I. polymerase
panel shows SDS-PAGE analysis of whole cell extracts of appropriate overproducer ,sniarts
(t before induction = sampled )0 treatment. The after times the at and the inducing indicated
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 products. respective expressed