From the
Wild-type DNA polymerase II (pol II) and an
exonuclease-deficient pol II mutant (D155A/E157A) have been
overexpressed and purified in high yield from Escherichia
coli. Wild-type pol II exhibits a high proofreading 3`-exonuclease
to polymerase ratio, similar in magnitude to that observed for
bacteriophage T4 DNA polymerase. While copying a 250-nucleotide region
of the lacZ
Three DNA polymerases have been identified in Escherichia
coli(1) . Pol I
As a consequence of these
newly-established pol II phenotypes(10, 12) ,
E. coli JM109 cells carrying the polB gene
under Ptac control on a high copy number plasmid were used to
overproduce wild-type pol II and the 3`-exonuclease-deficient pol II
derivative (pol II ex1). A typical preparative scale purification
starting with 300 g of dry cells yielded about 300 mg of purified pol
II (). Approximately 75-80% of the enzyme activity
was recovered with a corresponding 35-40-fold purification (). The degree of purification and recovery of polymerase
activity for the exonuclease-deficient enzyme was similar to wild-type
pol II (data not shown). Fractions from each step of the purification
were analyzed by SDS-polyacrylamide gel electrophoresis and visualized
by silver staining (Fig. 1). Each enzyme preparation appeared to
be at least 95% chromatographically pure. Crystals suitable for high
resolution x-ray diffraction have been obtained from the fraction IV
material for the wild-type protein(25) . The specific activity
of wild-type pol II was routinely 3-fold higher than for the pol II ex1
enzyme suggesting that the Asp
In
order to determine the nature of the errors generated by pol II ex1 and
to determine quantitative error rates and the contribution of the
exonuclease to fidelity, collections of independent lacZ mutants from
reactions containing 50 µM dNTPs were analyzed by DNA
sequence analysis. The data are summarized in I, and the
distribution of mutants containing single base substitutions and
frameshifts are shown in Fig. 6. Of 54 mutants from reactions
catalyzed by wild-type pol II, 33 contained a single C
Although DNA polymerase II was discovered in 1970 (3) its precise role in DNA replication or repair has been
difficult to establish. Pol II contains both 5`-polymerase and 3`-
exonuclease activities (1) on a single 89.9-kDa polypeptide
chain(4, 5, 32) . It has been shown that pol II
can bypass abasic lesions in vitro(20) and is
regulated by the LexA repressor (4, 5, 20) as
part of the SOS regulon of E. coli(33) . Recent
experiments by Tessman and Kennedy (12) suggest that pol II may be
required for bypassing abasic lesions in vivo under conditions
where heat shock proteins are not induced. We have presented evidence
for the involvement of pol II in the repair of or tolerance to
oxidative damage and in influencing the rate of adaptive
mutation(10) .
A purification scheme has been presented, which makes use of
an overexpression system for wild-type pol II and exonuclease-deficient
enzyme, that can be carried out rapidly, yielding large quantities of
highly purified polymerase that has been used to prepare single
crystals suitable for x-ray diffraction analysis(25) . Based on
an active site titration of our preparations (Fig. 2), we have
determined that at least 50% of the purified wild-type and
exonuclease-deficient pol II molecules were active in extending
primer/template DNA in the presence of a DNA-heparin trap. This 50%
value is a minimum estimate of the fraction of active enzymes molecules
since the assay does not detect those enzymes that dissociated from the
primer/template DNA before incorporating a deoxynucleotide.
The
specific activity of our most highly purified pol II fraction (Fraction
IV) was calculated to be 1.8
Bacteriophage T4 polymerase contains a highly
active 3`-exonuclease proofreading activity(26) . Pol II and T4
pol have been classified as group B ``
Pol II
appeared to degrade single-stranded DNA about 1.5-fold more rapidly
than T4 pol and 400-500-fold more rapidly than KF, when
equivalent polymerase units were used for each enzyme (Fig. 3).
The relative 3`-exonuclease activities for the three polymerases were
also determined under DNA synthesizing conditions by measuring the
turnover of [
A pol II exonuclease-deficient (pol II ex1)
derivative was constructed by introducing two amino acid changes
(D155A/E157A) analogous to those made for a proofreading-deficient
mutant of bacteriophage T4 (D112A/E114A). The exonuclease-deficient
mutant pol II exhibited about a 1000-fold reduction in exonuclease
activity compared to wild-type pol II (Fig. 5), and a
3-4-fold reduction in polymerase activity was also observed. The
residual exonuclease activity that represents 0.1% of the normal level
may be due to contaminant wild-type DNA polymerase II encoded by the
chromosome.
Wild-type pol II is highly accurate, having base
substitution and frameshift error rates of
An unexpected and interesting result from the present study was the
frequent generation of errors involving loss of a large number of
nucleotides flanked by directly repeated sequences (Fig. 7). The
simplest model to account for these involves pol II synthesis through
the first repeat, disruption of repetitive terminal base pairs,
formation of base pairs involving the newly-made DNA and the downstream
direct repeat, and continued synthesis. Deletions consistent with this
model have been found in previous studies of the fidelity of several
different exonuclease-deficient DNA polymerases (reviewed in Refs. 40
and 41). However, here they are even observed above background mutant
frequencies in the lacZ mutant collection from the
exonuclease-proficient pol II reaction (Fig. 7, top). In
7 of 8 such mutants, perfect direct repeat homology involving 5, 7, or
8 base pairs was present at the deletion end points, with the eighth
example being homologous for 9 of 10 base pairs (if one G
The frequency of
deletions between direct repeats is substantially higher in the
exonuclease-deficient pol II mutant collection (I) and,
among those observed, several may involve imperfect direct repeats (Fig. 7, bottom). Intermediates involving imperfect
repeats would contain mismatches in the double-stranded template/primer
region that could slow polymerization and lead to proofreading by the
wild-type enzyme. The frequency and specificity data thus suggest that
some direct repeat-dependent polymerase errors are in fact proofread.
It is even possible that the absence of exonuclease activity may
promote such deletion errors, by a mechanism wherein unedited
nucleotide misinsertion generates a terminus that frays and realigns
with a downstream sequence that is more homologous by virtue of the
original misinsertion. This idea is supported by numerous observations
suggesting that one-base deletions are initiated by misinsertion
followed by realignment with a downstream sequence (for review, see
Ref. 40).
Our results demonstrate that the 3`-proofreading
exonuclease of DNA polymerase II plays an important role in rectifying
replication errors that can lead to frameshifts and deletions in
vitro. Recently we have obtained evidence that strains deficient
in the exonuclease function of pol II show a mutator phenotype in
nondividing cells.
Cells were induced with
isopropyl-1-thio-
Reactions (25 µl) contained
20 mM Tris-HCl buffer, pH 7.5, 2 mM dithiothreitol,
10 mM MgCl
gene, the fidelity of wild-type pol II is
high, with error rates for single-base substitution and frameshift
errors being
10
. In contrast, the pol II
exonuclease-deficient mutant generated a variety of base substitution
and single base frameshift errors, as well as deletions between both
perfect and imperfect directly repeated sequences separated by a few to
hundreds of nucleotides. Error rates for the pol II
exonuclease-deficient mutant were from
13- to
240-fold higher
than for wild-type pol II, depending on the type of error considered.
These data suggest that from 90 to >99% of base substitutions,
frameshifts, and large deletions are efficiently proofread by the
enzyme. The results of these experiments together with recent in
vivo studies suggest an important role for pol II in the fidelity
of DNA synthesis in cells.
(
)is involved in
repair of damaged DNA and processing RNA-primed Okazaki
fragments(1) , while pol III is required for replication of the
bacterial genome(2) . Although pol II was discovered more than
24 years ago(3) , its role in either replication or repair has
been difficult to define. It has been shown that pol II expression is
regulated by the lexA repressor, as part of the SOS regulon, where the
locus of the structural gene for pol II is coincident with the DNA
damage-inducible dinA gene(4, 5) . In vitro studies have established that DNA pol II activity (6, 7) and processivity (8) are strongly
stimulated by DNA pol III accessory proteins suggesting that the two
polymerases might share overlapping roles in the cell. An E. coli strain containing a deletion in the pol II structural gene
exhibited an increase in adaptive mutation rate(9) , suggesting
a role for pol II in localized DNA synthesis or repair in nondividing
cells(10) . Recently, we have shown that replacement of
wild-type polB with an exonuclease-deficient polB allele increases the rate adaptive mutation in E.
coli.
(
)
biochemical investigations of pol
II(7, 8, 13, 14) are especially timely
and important to pursue. Pol II is classified as a type II, human
-like polymerase (4) having considerable sequence
similarity to bacteriophage T4 pol. The enzyme contains both polymerase
and 3`-exonuclease activities within a single 89.9-kDa polypeptide
chain(4, 15, 16) . In this paper, we describe
the purification and biochemical properties of wild-type pol II and an
exonuclease-deficient pol II derivative (pol II ex1).
Chemicals
All nonradioactive dNTP substrates
were purchased from Pharmacia LKB Biotechnology Inc. Radioactive
[-
P]ATP (4000 Ci/mmol),
[
-
P]dNTP (4000 Ci/mmol), and
[methyl-
H]TTP (20 Ci/mmol) were
purchased from ICN Radiochemicals, Inc. (Irvine, CA).
Diethylaminoethyl-cellulose (DE52) and phosphocellulose (P-11) resins
were obtained from Whatman BioSystems Inc.
Enzymes
Phage T4 DNA polymerase (3.5
10
units/mg(17) , 1 unit is that amount of enzyme
required to incorporate 1 pmol of labeled dTMP into nucleic acid
product in 1 min at 37 °C) was kindly provided by Dr. L.
Reha-Krantz (University of Alberta, Canada). A measurement of T4 pol
specific activity was also carried out at 30 °C for direct
comparison with published data; the value obtained, 2
10
units/mg is consistent with the data in Ref.(17) . Klenow
fragment (7000 units/mg) was purchased from Pharmacia LKB Biotechnology
Inc. Pol II wild-type and a 3`-exonuclease-deficient pol II derivative
were purified as described below.
Primer/Template
Single-stranded M13 DNA template
was prepared as described(18) . Primers were synthesized using
an Applied Biosciences DNA synthesizer by Lynn Williams (Kenneth Norris
Jr. Comprehensive Cancer Center, University of Southern California, Los
Angeles) and used after purification by polyacrylamide gel
electrophoresis. Primer sequence p5, 5`-ATTAAATCCTTTGCCCG-3`, is
complementary to the numbered positions on wild-type M13 strand:
4640-4657. Primer p15C (5`-ATGATTAAGACTCCTTA-3`) can be annealed
to template TGG-1 (3`-TACTAATTCTGAGGAATGGTGAGTCATACAATCGT-5`). The
sequence of primer Prsp2 is 5`-GATCAGTCCTGTACGGTACTGACTGACC-3`.
Bacteria Strain and Plasmid
E. coli JM109 (recA1 (lac-proAB
) F`(traD36 proAB
lacI
lacZ
M15)) was kindly provided by Dr. Miriam Susskind,
University of Southern California. Plasmid pPROK-1 was purchased from
CLONTECH Laboratories, Inc. (Palo Alto, CA).
Construction of Pol II Overproducing Plasmid (pHY400) and
a Pol II 3`-Exonuclease Mutant (D155A/E157A) Overproducing Plasmid
(pHC700)
A 2.4-kb DNA fragment containing the dinA/polB(
)open reading frame was
obtained from plasmid pSH100 by polymerase chain reaction amplification
of the polB coding region(4) . The original
``inefficient'' GTG translation initiation codon was changed
to ATG using a polymerase chain reaction primer containing the
appropriate base change. This 2.4-kb polymerase chain reaction fragment
was subcloned into the pPROK-1 vector (a 4.6-kb plasmid vector
containing a Ptac promotor) to give a 7.0-kb plasmid construct, pHY400.
The sequence of polB was confirmed by DNA sequence analysis.
The expression of polB is under the control of the Ptac
promotor that is regulated by Lac I
. The polB gene
containing substitutions D155A/E157A was engineered using standard
oligonucleotide-directed mutagenesis procedures(19) . Mutations
in the plasmid were screened initially by restriction endonuclease
mapping (the mutant oligonucleotide encoding the alanine substitution
introduced a new restriction site for AflII endonuclease) and
later by DNA sequencing of the polB gene. A 2.4-kb fragment
corresponding to the polB open reading frame containing the
desired mutations was inserted into the pPROK-1 vector resulting in a
7.0-kb plasmid, pHC700.
Cell Growth and Cell Lysis
E. coli JM109
cells carrying the polB gene on the overproducing plasmid
pHY400 (wild-type pol II) or pHC700 (3`-exonuclease-deficient pol II, i.e. pol II ex1) were grown in LB with 50 µg/ml ampicillin
in a 180-liter fermenter at 37 °C. The overproduction of pol II
protein was induced by adding isopropyl
-D-thiogalactoside to the cells at midlog phase (A
about 0.8) to a final concentration of 0.4
mM. Two hours after isopropyl
-D-thiogalactoside
induction, cells were harvested and resuspended in a volume of storage
buffer (sterile 50 mM Tris-HCl, pH 7.5, 10% sucrose) equal to
the wet weight of the cells in grams. Cells were rapidly frozen by
resuspension in liquid nitrogen and were stored at -70 °C.
Lysis buffer (50 mM Tris-HCl, pH 7.5, 10% sucrose, 0.1 M NaCl, 15 mM spermidine) was added to frozen cells to
achieve a final concentration of 0.2 g of cells/ml. Cells were thawed
at 4 °C and the pH was adjusted to 7.7 with 2 M Tris base.
Lysozyme was added to a final concentration of 0.2 mg/ml. The cell
slurry was incubated for 1 h at 4 °C, 4 min at 37 °C, and
centrifuged at 11,800 rpm for 1 h in a Sorvall GSA rotor. The
supernatant (Fraction I) was decanted and kept on ice.
Purification of Pol II
Ammonium sulfate was added
slowly to Fraction I, to a final concentration of 30% (w/v), and the
suspension was kept at 4 °C overnight, without stirring. The
suspension was centrifuged in a Sorvall GSA rotor (11,800 rpm, 40 min)
and the supernatant was discarded. A volume of PC/25 buffer (50 mM Tris-HCl, pH 7.5, 20% glycerol, 1 mM EDTA, 5 mM
dithiothreitol, 25 mM NaCl) equal to one-fifth to one-tenth of
the original volume of Fraction I was added to the ammonium sulfate
pellet and the protein was gently resuspended (Fraction II). Fraction
II was dialyzed against PC/25 buffer until the conductivity reached a
value equivalent to that of 40 mM NaCl. After dialysis,
Fraction II was diluted with PC/25 buffer to a protein concentration of
approximately 10 mg/ml and then loaded onto a P-11 phosphocellulose
column, 5 70 cm (inner diameter
length). The column was
washed with 1 column volume of PC/25 buffer and an additional column
volume of PC/200 buffer (the same components as buffer PC/25 except
that the NaCl concentration was 200 mM) to elute DNA
polymerase III. Pol II protein was eluted with a gradient of
200-500 mM NaCl in buffer PC (8 column volumes). The pol
II fractions (eluting between 225 and 250 mM NaCl) were pooled
to give Fraction III. Fraction III was dialyzed against PK20 buffer (20
mM potassium phosphate, pH 6.8, 15% glycerol, 1 mM EDTA, 5 mM dithiothreitol) until the conductivity reached
that of PK30 buffer (30 mM potassium phosphate, pH 6.8) and
loaded on a DEAE column, 5
70 cm (inner diameter
length). The DEAE column was washed with 2-column volumes of PK20
followed by elution with an 8-column volume gradient of 20-350
mM potassium phosphate (PK20 to PK350). The pol II fractions
(typically eluting at 100-140 mM potassium phosphate)
were pooled as Fraction IV and stored at -70 °C. The
procedure used to purify the exonuclease deficient pol II mutant was
the same used for wild-type pol II. The specific activity and recovery
at each step of purification for wild-type pol II is contained in .
Deoxyribonucleotide Incorporation Assay
The
deoxynucleotide incorporation activity of pol II was measured using
[methyl-H]TTP and activated salmon sperm
DNA as described(20, 21) . Reactions contained 2.5
mM dithiothreitol, 20 mM Tris-HCl, pH 7.5, 7.3 mM MgCl
, 6 mM spermidine/HCl, 1 mg/ml bovine
serum albumin, 1.1 mM activated DNA, 60 µM dATP,
dCTP, dTTP, dGTP, and [
H]dTTP. One unit of enzyme
catalyzed the incorporation of 1 pmol of [
H]TMP
into acid insoluble material in 1 min at 37 °C. T4 pol
deoxynucleotide incorporation activity was measured by the same method
except the reaction buffer contained 67 mM Tris-HCl, pH 8.8,
16.7 mM (NH
)
SO
, 0.5 mM dithiothreitol, 6.7 mM MgCl
, and 167
µg/ml bovine serum albumin.
Protein Assay
Protein concentration was measured
by the Bradford assay using materials and protocol supplied by Bio-Rad.
Silver staining of protein gels were carried out using protocols and
materials supplied in the ICN Rapid Ag-Staining Kit.
Active Site Titration of Purified Pol II Protein Using
DNA-Heparin Trap
5` End-labeling of primer p15C was carried out
in a labeling reaction (20 µl) containing 50 mM Tris-HCl,
pH 7.5, 2 mM MgCl, and 0.5 mM dithiothreitol, 3.0 µM primer, 0.3 µM [
-
P]ATP, and 10 units of T4
polynucleotide kinase. The labeling reaction was performed at 37 °C
for 1 h and terminated by heating it to 90 °C for 5 min. 2 µl
of 100 mM EDTA (dissolved in 5 mM Tris-HCl, pH 8.0)
was added to the labeling reaction to chelate Mg
ions. Template TGG-1 was added to the above solution and
conditions were adjusted to attain 17 mM Tris-HCl, pH 7.5, 0.7
mM MgCl
, 0.17 mM dithiothreitol, 1.0
µM primer, 1.25 µM template TGG-1, and 3
mM EDTA. Annealing was carried out by heating the annealing
reaction mixture to 90 °C for 3 min and then cooling to room
temperature over a 1-h time period. Under these conditions, more than
96% of annealed primer could be utilized by polymerase as judged by the
primer extension experiment described below (data not shown). A
combination of 0.1 mg/ml heparin sulfate and 0.66 mg/ml calf thymus
DNA, prepared as described (22) was completely effective in
trapping free (i.e. dissociated) pol II. For a typical
reaction (20 µl), 4 µl of enzyme was preincubated with 8 µl
of primer/template/dNTP mixture for 1 min at 37 °C to form
enzyme-primer-template complexes, and primer extension was initiated
with 8 µl of MgCl
/trap mixture. The final
concentrations were 3-50 nM polymerase, 50 mM Tris-HCl, pH 7.5, 50 nM primer/template DNA, 50 µg/ml
bovine serum albumin, 0.5 mM dithiothreitol, 60
µM dNTPs, 7.3 mM MgCl
, 0.1 mg/ml
heparin sulfate, and 0.66 mg/ml calf thymus DNA. Following incubation,
the reaction was quenched by addition of 20 mM EDTA (40
µl) in 95% formamide. The efficiency of the trap was determined by
preincubating pol II with the trapping mixture before addition of
labeled primer/template. Separation of
P-labeled primers
of different lengths was carried out by gel electrophoresis, and the
fraction of primer molecules extended by addition of 1 or more
nucleotides was determined using a PhosphorImager (Molecular Dynamics).
The amount of primer extended represents the minimum fraction
of active enzyme-primer-template complex and therefore determines a
lower limit for the fraction of active pol II (see
``Results''). The minimum fraction of active pol II is given
by the slope of the curve obtained by plotting the amount of primer
extended (nM) versus the amount of enzyme
(nM) in the reaction.
Measurement of Exonuclease Activity by Primer
Degradation
5` End-labeling and gel electrophoresis were carried
out as described(23) . To measure 3`-exonuclease activity, 20
µl total reaction volume, 50 nM primer p5 (as
single-stranded DNA substrate) was incubated with pol II, T4 pol, or
KF. The concentrations of the three enzymes were adjusted to an
equivalent of 0.04 pol II units. Incubations were carried out for
different times at 37 °C and reactions were terminated as described
above. Reaction buffers for pol II and KF contained 50 mM Tris-HCl, pH 7.5, 50 µg/ml bovine serum albumin, 0.5 mM dithiothreitol, and 7.3 mM MgCl. The reaction
buffer for T4 pol was given above. The rate of primer degradation was
determined from the slope of the linear region of a plot of percent
primer degraded versus time. A comparison of the exonuclease
activities of wild-type pol II and pol II ex1 (D155A/E157A)
was carried out by the same method, except that primer Prsp2 was used
as the substrate (28-mer, at a concentration of 180 nM) and
0.4 units of wild-type pol II (or 4 units of pol II ex1) was present in
the reaction mixture (40 µl total volume).
Turnover Assay
Primer/template p5-M13 was used for
measuring dNTP dNMP turnover during DNA synthesis. The reaction
conditions were the same as those used to measure 3`-exonuclease
activity by primer degradation except the primer p5 was unlabeled and
[
-
P]dCTP or
[
-
P]dTTP (4000 Ci/mmol, 3.3
µM) and 95 µM dATP were used to allow primers
to be extended by addition of 3 nucleotides. 0.4 pol II units were used
to measure turnover of the correctly inserted nucleotide (dCTP), and
1.2 pol II units were used to measure turnover of the incorrectly
inserted nucleotide (dTTP). The concentrations of T4 pol and KF were
adjusted to have the same number of polymerase units that was used for
pol II, i.e. the nucleotide insertion rates were adjusted to
be the same for all three enzymes. Product dNMP was separated from
unreacted dNTP and contaminating dNDP by electrophoresis on an 8%
polyacrylamide gel for 1 h at 2000 V. The rate of turnover was
determined from the slope of the linear region of a plot of the amount
of dNMP formed versus time. Contaminating levels of
radioactive dNDP in dNTP substrates were typically about 0.5-1%
and were easily resolved from the product dNMP (see, e.g.Fig. 4); levels of contaminating dNMP in freshly prepared
substates were negligible compared to the amount of product formed
during the reaction.
Figure 4:
Comparison of pol II, T4 pol, and KF dNTP
dNMP turnover activities. A, incorporation and excision
of a correct deoxynucleotide [
-
P]dCTP
opposite template G on the unlabeled primer/template DNA shown at the
top of the figure. Incorporation of labeled dCTP is indicated by the
location of the extended primer band corresponding, primarily, to the
addition of three nucleotides. Turnover of the dCTP substrate is
indicated by the dCMP radioactive spot. The heavy radioactive spot
migrating at the bottom of the gel corresponds to unreacted dCTP, and
the spot migrating between the unreacted dCTP and dCMP product is dCDP
contaminant present in the dCTP substrate. B, excision of an
incorrectly inserted deoxynucleotide
[
-
P]dTTP opposite template G on the
unlabeled primer/template shown at the top of the figure. No
stable incorporation of dTTP is observed as shown by the absence of a
detectable radioactive band at the location denoted by ``extended
primer.'' Turnover of the dTTP substrate is indicated by the dTMP
radioactive spot. The radioactive spot migrating at the bottom of the gel is unreacted dTTP, and the spot located between dTTP
and dTMP product is dTDP contaminant present in dTTP. Reactions using
pol II, T4 pol, and KF were carried out for the indicated times. The
position of the unextended primer (5`-
P-labeled 17-mer) is
shown in the left-hand lane of each gel. The concentrations of
the three enzymes used for turnover of the correct nucleotide (dCMP)
were adjusted to have the same number of polymerase units, equivalent
to 0.4 pol II units. Three-fold higher concentrations of each
polymerase (1.2 pol II units) were used to measure turnover of the
incorrect nucleotide (dTMP).
Forward Mutagenesis Assay
The assay, described in
detail in Ref. 24, measures errors in the wild-type lacZ gene of M13 mp2. Correct polymerization during synthesis to fill a
390-base single-stranded gap produces DNA that yields dark blue M13
plaques upon transfection of an appropriate E. coli host
strain. A wide variety of errors at many different sites are scored as
lighter blue or colorless plaques. After sequence analysis of a
collection of independent mutants, errors rates per detectable
nucleotide incorporated were calculated as described(24) . The
DNA polymerase reaction conditions and description of product analysis
are in the legend to . E. coli strains,
bacteriophage M13 mp2, and the sources of materials used for the M13
mp2 fidelity assay have been described(24) .
to Ala and Glu
to Ala mutations in the exonuclease domain, that reduce levels of
exonuclease to 1/1000th the level of wild-type, appeared to cause a
small reduction in the polymerization rate of the enzyme.
Figure 1:
Silver-stained polyacrylamide gel
showing protein bands during purification of E. coli wild-type
and exonuclease-deficient (exo) pol II. Lane 1,
prestained molecular weight markers; lane 2, F1 crude lysate
(17 µg total protein); lane 3, F2 ammonium sulfate
fraction (20 µg total protein); lane 4, phosphocellulose
column fraction (5 µg total protein); lane 5,
DEAE-cellulose column fraction (5 µg total protein). The
purification procedure is described under ``Experimental
Procedures.'' The specific activity and recovery at each stage of
purification for the wild-type pol II is given in Table
I.
A
titration using increasing levels of pol II at a constant
primer/template DNA concentration was carried out to determine the
fraction of pol II (fraction IV) that was active (Fig. 2A). Pol II was allowed to bind to primer/template
DNA and extend a P-labeled primer molecule, at most once,
by carrying out the reaction in the presence of excess unlabeled DNA
and heparin to trap free polymerase molecules (``Experimental
Procedures''). The primer extension reaction in the absence of
trap is shown in lane 1, where pol II was able to engage and
elongate primer/template DNA multiple times. The effectiveness of the
trap is seen in lane 8, where the addition of trap prior to
initiation of the polymerization reaction, by addition of
Mg
, was successful in eliminating detectable
elongation of
P-labeled primer strands. Increased levels
of pol II resulted in increased primer utilization (lanes
3-7), and the fraction of primer/template DNA extended is
plotted as a function of pol II concentration in Fig. 2B. From the slope of the line we conclude that at least 50% of the pol II molecules were active. The spots
corresponding to unelongated primers (P) are overexposed (Fig. 2A), but we have verified by integrating all of
the bands on the gel that the disappearance of intensity in the P band
is converted quantitatively into primer extension bands. Since not all
polymerase binding events are productive, i.e. a bound pol
II-DNA complex might dissociate prior to elongation followed by
trapping of the dissociated enzyme, the observed molar ratio of
extended DNA/pol II (
0.5) represents a lower limit for the
fraction of active pol II (Fig. 2B). The same criteria
was used to verify that at least half of the purified
exonuclease-deficient pol II ex1 (D155A/E157A) molecules were active
(data not shown).
Figure 2:
Active
site titration of pol II. A, lane 1, primer extension
was carried out using 2.5 nM pol II in the absence of
DNA-heparin trap (see ``Experimental Procedures''); lane
2, 46 nM pol II incubated with labeled primer/template
DNA and all four dNTP substrates; lane 3, primer extension was
carried out using 3 nM pol II in the presence of a DNA-heparin
trap; lanes 4-7, same as lane 3 using pol II at
6, 12, 24, 46 nM, respectively; lane 8, 46 nM pol II was preincubated with DNA-heparin trap and all four dNTP
substrates and the reaction was then initiated by addition of
Mg. The primer/template DNA was present at 50
nM. Lane 2 serves as a control showing that trace
amounts of Mg
present in the primer/template DNA
solution are insufficient to cause measurable primer elongation. Lane 8 is a control demonstrating the effectiveness of the
DNA-heparin trap; preincubation with trap eliminates elongation of
5`-
P-labeled primer molecules. B, plot showing
the amount of active pol II-DNA complex (given by the amount of primer
extended) versus the amount of pol II present in the reaction.
The minimum fraction of active purified pol II, given by the
slope of the line, is equal to 50%.
A Comparison of Exonuclease Activities of Pol II, T4 Pol, and
Pol I(KF)
It was of interest to compare the polymerase and
3`-exonuclease proofreading activities of pol II and bacteriophage T4
pol because both enzymes are class B polymerases (human -like
polymerases) sharing five highly conserved sequence motifs(4) .
Wild-type T4 pol has an exceptionally high 3`-exonuclease activity and
is able to excise 10-20% of correctly paired
nucleotides(26, 27, 28) , depending on the
identity of the the base pair and sequence context (29). Equal
polymerase units of pol II, T4 pol, and KF were incubated in the
presence of a 5`-
P-labeled single-stranded 17-mer without
dNTPs (Fig. 3). Degradation of the 17-mer occurred more rapidly
for pol II than T4 pol, with full-length input primer disappearing
within 1 min of incubation at 37 °C. In the case of KF, the primer
remained essentially intact for incubations of up to 10 min.
Degradation rates were determined to be 6.9
10
mol/min, 4.7
10
mol/min, and 1.5
10
mol/min for pol II, T4 pol, and KF,
respectively.
Figure 3:
Comparison of pol II, T4 pol, and KF
3`-exonuclease activities on single-stranded DNA. A
5`-P-labeled single-stranded DNA oligonucleotide (17-mer)
was incubated with pol II, T4 pol, and KF for the reaction times shown.
The concentrations of the three enzymes were adjusted to have the same
number of polymerase units, equivalent to 0.04 pol II units. The first lane on the left-hand side indicates the
position of undegraded 5`-
P-labeled single-stranded
17-mer.
A measurement of 3`-exonuclease activity under
synthesizing conditions (dNTP dNMP turnover), was carried out
for insertion of a correct (dCTP, Fig. 4A) and an
incorrect (dTTP, Fig. 4B) deoxynucleotide opposite G by
pol II, T4 pol, and KF. An increase in dNTP
dNMP turnover as a
function of incubation time was observed for each enzyme. Using equal
polymerase units, turnover of both correct and incorrect inserted
dNMP's were slightly larger for pol II than T4 pol, with both
polymerases exhibiting far greater proofreading activity than KF. The
radioactive spot migrating in between the dense spot of unreacted
-
P-labeled dNTP substrate and product dNMP is dNDP,
which likely resulted from the spontaneous hydrolysis of dNTP. The
level of dNDP is about 0.5% of the input dNTP and remained constant
during the course of the reaction. A small amount of stable correct
incorporation of dCMP was observed as an extended primer band (Fig. 4A), but there was no detectable band
corresponding to the stable misincorporation of dTMP (Fig. 4B). Turnover rates of the correct nucleotide
(dCTP) were about 6.7
10
mol/min, 5.6
10
mol/min, and 1.1
10
mol/min for pol II, T4 pol, and KF, respectively (Fig. 4A). For the incorrect nucleotide (dTTP), turnover
rates were 5.6
10
mol/min, 2.3
10
, and 1.5
10
for pol
II, T4 pol, and KF, respectively (Fig. 4B). The rates of
turnover of incorrect nucleotide per polymerase unit were less than
correct nucleotide because dNTP
dNMP turnover measures insertion
followed by excision, and insertion of incorrect nucleotides occur much
less frequently than correct nucleotides. For these experiments, a
3-fold higher concentration of each of the polymerases was used for
measurements of incorrect compared to correct turnover.
Exonucleolytic Degradation of Single-stranded
DNA
We compared the ability of wild-type pol II and the pol II
ex1 mutant (D155A/E157A) to degrade single-stranded DNA (Fig. 5).
During incubations with equivalent polymerase units, degradation of a
28-mer was detected within a 5-s incubation with the wild-type enzyme,
but was just barely detectable after 80 s with the
exonuclease-deficient mutant (Fig. 5). The residual level of
3`-exonuclease activity in the mutant enzyme was calculated to be
approximately 0.1% of the wild-type activity, based on the fraction of
28-mer remaining intact.
Figure 5:
Comparison of 3`-exonuclease activities of
wild-type pol II and an exonuclease-deficient (exo)
pol II mutant (D155A/E157A). A, wild-type pol II (0.4
polymerase units) and B, pol II exo
mutant
(4 polymerase units) were used to hydrolyze a single-stranded
5`-
P-labeled single-stranded DNA oligomer as described
under ``Experimental Procedures.'' The DNA oligomer (28-mer)
was present at a concentration of 180
nM.
Fidelity of Wild-type and Exonuclease-deficient Forms of
Pol II
The fidelity of both pol II forms was determined during
synthesis to fill a 390-nucleotide single-stranded gap containing the lacZ complementation reporter gene sequence. Gap-filling
synthesis by the wild-type polymerase in a reaction containing 50
µM dNTPs generated products that had a lacZ mutant
frequency that was elevated about 2-fold compared to an uncopied
control DNA substrate (). When a wild-type pol II reaction
was performed using 1 mM dNTPs, the mutant frequency increased
to a value more than 10-fold higher than the uncopied control value.
This increase is consistent with stimulation of polymerization at the
expense of exonucleolytic proofreading(28, 30) . The
2-fold higher mutant frequency observed at 1 mM dNTPs is
consistent with a dNTP-dependent increase in extension of mispaired and
misaligned intermediates(31) . In comparison, pol II ex1
generated substantially higher lacZ mutant frequencies than did the
wild-type enzyme at both dNTP concentrations ().
T
transition mutation. These mutants, which account for most of the
increase in mutant frequency above the control value ()
are likely the result of cytosine deamination, with wild-type pol II
``correctly'' inserting dAMP opposite template U residues.
Most of the other classes of mutants from the wild-type pol II reaction
occurred at background frequencies, suggesting that they were not the
result of synthesis errors in vitro. The frequency data were
used to calculate (see legend to I) ``less than or
equal to'' error rates per detectable nucleotide polymerized, for
the single base errors and the two nucleotide deletions.
Figure 6:
Spectra of single-base errors by E.
coli DNA polymerase II. Three lines of primary DNA sequence for
the lacZ -complementation gene in M13 mp2 are shown. The sequence
is that of the viral (+) template strand. Position +1 is the
first transcribed base. The spectrum above the lines of
sequence is of mutants generated by wild-type enzyme, while that below the lines of sequence is of mutants generated by the
exonuclease-deficient polymerase (pol II ex1). In both cases the
reactions contained 50 µM of each dNTP. Substitutions are
indicated by a single letter and are the new base found in the viral
DNA. For frameshifts, the loss of a base is indicated by a
, while
the addition of a base is indicated by an
. When frameshifts
occur at iterated nucleotides, it is not possible to distinguish which
base was lost or added.
The only
mutants observed in the wild-type pol II collection having a frequency
above that of the uncopied control values were the eight deletions of
more than two nucleotides (Fig. 7, top). None of these
mutants has been observed previously in sequence analysis of over 200
mutants from uncopied controls, suggesting that they are errors
generated by wild-type pol II. These mutants had lost between 49 and
268 nucleotides and in each instance the deletion end points were
flanked by short repeated sequences. One particular mutation, a
182-nucleotide deletion between a perfect 7-base pair direct repeat,
was recovered four times in this small collection. Only one of the
eight mutants was flanked by perfect repeat shorter than 5 base pairs,
and even here the repeat homology is higher if formation of GT
pairs in the repeat is permitted (see legend to Fig. 7and
``Discussion'').
Figure 7:
Deletion mutations recovered from
reactions with wild-type and exonuclease-deficient pol II. Shown are
lacZ mutants recovered from pol II reactions wherein 22 or more
nucleotides have been deleted. Nucleotide +1 is the first
transcribed nucleotide of the gene. The deletion end points are
indicated by slashes, with the number of nucleotides deleted
as listed and the homology at the deletion end points indicated in the
last column. For example, the first deletion listed for wild-type pol
II had lost 268 nucleotides, from nucleotide -126 through
nucleotide 142, and a perfect 8-base pair direct repeat sequence was
present at the deletion end points. Complementarity at the end points
is indicated by capital letters, while non-complementary
nucleotides are indicated by lower case letters. Underlined lower
case letters indicate positions where a GT or T
G
mispair would be located in a putative misaligned intermediate formed
during synthesis proceeding from right to left using the template
strand shown. Numbers in parentheses under
``Homology'' indicate homologies obtained by including the
involvement of these two types of mispairs. Numbers in parentheses under ``Bases Lost'' indicate the number
of times the particular mutation was
recovered.
The collection of mutants from the pol II
ex1 reaction was much more diverse, containing a variety of single base
substitutions and frameshifts and deletions of two or more nucleotides (I, Figs. 6 and 7). Rates calculated for the single-base
substitution and frameshift errors and two-base deletions are higher by
values ranging from 13-fold to
240-fold for exo-pol II than for
wild-type pol II (I), consistent with a role for the
exonuclease in proofreading each of these classes of errors. Among
deletions involving the loss of more than two bases, 10 had lost more
than 20 nucleotides, and each of these had substantial but often
imperfect homology at the deletion end points (Fig. 7, bottom). The others (not shown) had lost fewer than 10
nucleotides and most had limited homology at the end points.
The purification and properties of
wild-type pol II and an exonuclease-deficient pol II mutant (pol II
ex1) provide additional insights into the role of this polymerase in
cells.
10
units/mg. Recently,
another laboratory has reported a specific activity for purified DNA
polymerase II which is nearly 11,000 times larger(13) . This
reported value is undoubtedly in error since their steady state rate
exceeds the fastest measured presteady state synthesis rate by
4-5-fold(34) . Our specific activity measurements are
consistent with values for other DNA polymerases. As we have previously
reported, DNA polymerase II can be made more processive by the addition
of DNA polymerase III accessory factors which increase the specific
activity of pol II by preventing dissociation from the DNA
template(8) .
-like''
polymerases because of their similarity in sequence to five conserved
regions of eucaryotic pol
(4, 35) . It is of
considerable interest to compare the relative levels of
nuclease/polymerase activities for pol II and T4 pol, not only because
of their sequence similarities, but also because of their effects on
mutagenesis. Mutations in T4 pol that decreased nuclease-to-polymerase
ratios in vitro were shown to confer mutator phenotypes for
base substitution mutations in vivo and conversely mutants
that increased nuclease/polymerase ratios conferred antimutator
phenotypes(17, 26, 36, 37) .
-
P]dNTP to
[
-
P]dNMP (Fig. 4). The rates of
turnover of correctly inserted dCMP opposite G were roughly similar for
pol II and T4 pol, and about 60-fold greater than observed for KF (Fig. 4A). Turnover rates of misinserted dTMP opposite G
were similar for pol II and T4 and approximately 20-fold greater than
for KF (Fig. 4B). Thus pol II and T4 pol have
essentially the same ratio of nuclease/polymerase activities even
though the specific activity of T4 pol is approximately 100-fold
greater than pol II, which undoubtedly reflects its role as the
replicative polymerase.
10
(I). In contrast, the exonuclease-deficient
polymerase generated a variety of different errors during DNA synthesis in vitro. Assuming that the two amino acid substitutions in
the conserved exonuclease motif that are responsible for diminished
exonuclease activity do not affect the selectivity of the polymerase,
the differences in error rates between the two polymerases under
identical reaction conditions represent the contribution of
proofreading to fidelity for each type of error. The data suggest that
single base substitutions, single base additions, and a variety of
deletion errors are proofread by the highly active intrinsic 3`-
exonuclease activity of pol II. The substitutions likely result from
direct misinsertion followed by mispair extension by the polymerase,
while models involving misaligned template/primers initiated by strand
slippage (38) or base misinsertion (39) have been
presented that can account for the one- and two-base frameshift errors
observed at repetitive and non-reiterated sequences (I).
T pair
is allowed). This suggests that, if the degree of homology is
sufficient, deletions between direct repeats can be generated by
wild-type pol II despite the presence of a highly active exonuclease
that successfully proofreads point mutations.
These biochemical and genetic results
suggest a fundamental role for DNA polymerase II in maintaining the
sequence integrity of DNA.
Table: Purification of E. coli DNA polymerase II from
E. coli JM109
-D-galactopyranoside to overproduce pol
II.
Table: Fidelity of wild-type and exonuclease-deficient
forms of E. coli DNA polymerase II
, dATP, dTTP, dGTP, and dCTP as
indicated, 30 fmol of (150 ng) gapped M13mp2 DNA and 5-14 units
of DNA polymerase II. Reactions were incubated for 60 min at 37 °C
and terminated by adding EDTA to 15 mM. Analysis of 15 µl
of the reaction by agarose gel electrophoresis showed that all
reactions filled the gap to the extent that the DNA migrated coincident
with the fully double-stranded RFII standard (e.g. see Ref.
24). The mutant frequency of uncopied DNA ranges from 5.1 to 7.0
10
.
Table: Average error rates by class
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.