THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
VOl. 266, No. 12, Issue of April 25, pp. 7 W 7 8 9 2 , 1 9 9 1
Printed in U.S.A.
Specificity and Enzymatic Mechanism of
the Editing Exonuclease of
Escherichia coli DNA Polymerase 111"
(Received for publication, December 3, 1990)
Stephan BrenowitzS, Sunye KwackS, Myron F. Goodman$,Mike O'Donnellli, and Harrison EcholsS
From the $Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720, the
§Department ofBiological Sciences, University of Southern California,L o s Angeles, California 90089, and the VDepartment of
Microbiology, Cornell University Medical College, New York, New York10021
Exonucleolytic editing is a major contributor to the
fidelity of DNA replication by the multisubunit DNA
polymerase (pol) I11 holoenzyme. To investigate the
source of editing specificity, we have studied the isolated exonuclease subunit, t, and the pol I11 core subassembly, which carries the 6, 0 , and a (polymerase) subunits. Using oligonucleotides with specific terminal
mismatches, we have found that both t and pol 111 core
preferentially excise a mispaired 3' terminus and
therefore have intrinsic editing specificity. For both
and pol I11 core, exonuclease activity is much more
effectivewithsingle-strand
DNA; with adoublestrand DNA, the exonuclease is strongly temperaturedependent. We conclude that the e subunit of pol I11
holoenzyme is itself a specific editing exonuclease and
that the source of specificity is the greater melting
capacity of a mispaired 3' terminus.
Earlier studieswith a single mismatch indicated that both pol
111 core and isolated t have a preference for a mispaired 3'
terminus (8,12). In this work we have examined in more
detail the specificity and the mechanism of exonucleolytic
proofreading.
First, we wanted to determine if editing specificity is a
property of the exonuclease subunit alone. Based on the
crystal structure, the editing specificity of DNA polymerase I
appears not to be intrinsic to the exonuclease domain, but
depends on the transferof single-strand DNA from the polymerase domain tothe exonuclease domain (13, 14). The
exonuclease active site must function solely as a single-strand
exonuclease, because there is no room for duplex DNA (13,
15); therefore the exonuclease site makes no direct selection
for a mispaired base. Kinetic studies havesuggested that
editing by pol I is achieved mainly by a delay in elongation
from a mismatched primer terminus (16). Thus editing does
not result from the intrinsic specificity of the exonuclease,
but ratherdepends on a kinetic delay in polymerization, which
DNA replication is carried out with extremely high accu- allows more time for transfer of single-strand DNA into the
racy. Error frequencies during duplication of the Escherichia exonuclease site. If the editing mechanism suggested for pol I
coli genome are 10-9-10"0 per base replicated (1).To achieve was also used by pol 111, the isolated t subunit would not
this fidelity, a DNA polymerase must have an exceptional distinguish a correctly paired from a mispaired 3' terminus.
ability to discriminate against incorrect base pairs, which may In this work, we have examined the editing specificity of the
exhibit only slight structural and energetic differences from t subunit and pol I11 core with a series of correctly paired or
the correct base pairs. Fidelity is achieved by a polymerase in mispaired oligonucleotides annealed to bacteriophage M13
a two-step process: (1) base selection, correct selection of the DNA. These DNA substrates provided all 16 possible combicomplementary dNTP during 5' --$ 3' incorporation; (2) ed- nations of correct and incorrect base pairs at the primer 3'
iting,3' + 5' exonucleolytic excision of a noncomplementary terminus. We have found that both pol I11 core and t specifideoxynucleotide misinserted at the3' end of a growing DNA cally excise incorrectly paired 3' termini more rapidly than
chain.With the additionalcontribution of postreplicative correctly paired termini.
We also wanted to determine the feature of DNA structure
mismatch repair, the high fidelity of genome duplication is
that allows the exonuclease to recognize mispairs. There are
achieved (2-4).
DNA pol' I11 holoenzyme is the enzyme primarily respon- two plausible general mechanisms for selectivity in editing:
sible for chromosomal replication in E. coli and therefore is recognition of departures from the equivalent geometry of the
probably the major determinant of the fidelity of genome Watson-Crick base pairs,and melting capacity of the 3'
duplication. The pol I11 holoenzyme contains 10 distinct terminus. Geometric recognition has been implicated as the
t, 6, T, y, 6, 6', J., x, and p (5,6). The critical determinantfor the specificity of base selection in the
polypeptide subunits: CY,
polymerase reaction (4, 17, 18). In the melting model, the
t subunit, the dnaQ gene product, is the 3' + 5' proofreading
exonuclease (7,8). The CY subunit, the d n u E gene product, is exonuclease is intrinsically a single-strandnuclease that prefthe 5' + 3' polymerase (9, 10). The CY, 6, and t subunits erentially removes a misinserted base because the mismatch
compose pol I11 core, the smallest subassembly of pol I11 at the 3' end is more often in a single-strand configuration
prepared from the holoenzyme (11).By the use of isolated t (19, 20). In the case of pol I, Brutlag and Kornberg (19)
subunit and pol I11 core, we can study the mechanism of demonstrated that the exonuclease works best on singleediting in the presence and absence of the polymerase subunit. strand DNA and that thereis a marked increase in exonuclease activity with duplex DNA as temperature is increased.
* This work was supported by National Institutes of Health Grants These data suggest that melting of the 3' terminus is the
CA 41655,GM 21422, and GM 38831.The costs of publication of this primary determinant for editing. As noted above, structural
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement" in accord- studies with pol I have also supported a melting model for
editing by pol I.In this study, we have examined the effect of
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
temperature andstrand specificity on the exonuclease activity
The abbreviations used are: pol, polymerase; N, nucleoside.
7888
7889
Editing Specificity of E. coli DNA Pol 111
of pol I11 core and e; our data support the melting model for
editing by pol 111.
Finally, we have carried out a steady-state analysis of the
exonuclease activity of pol I11 core to determine the kinetic
parameters that provide editing specificity. We have found
that VmmX
discrimination is the primary kinetic property that
characterizes preferential excision of a misinserted base at
the 3' terminus.
EXPERIMENTAL PROCEDURES
Materials-Purified f subunit was prepared as described previously
(8). Purified core subassembly was prepared as described previously
(11). T4 polynucleotide kinase was purchased from New England
Biolabs. Oligonucleotide substrates and the 35-mer template were
synthesized by conventional solid phase methods. Single-strand M13
DNA was purified from E. coli strain JM103 using published procedures (21). [-pR'P]ATP (>5000 Ci/mmol, 10 mCi/ml) was purchased
from Amersham Corp.
Preparation of DNA Substrates-Sixteen oligonucleotide substrates were annealed to M13 DNA to generate the 16 possible correct
and incorrect base pairs. Oligonucleotides (20-mers) were synthesized
complementary to four unique M13 sites with a variable 3'-terminal
base ( G ,A, T, C). The M13 DNA coordinates were: N .C (3576-3595);
N .T (2223-2242); N. A (5386-5405); N. G (4637-4656). Oligonucleotide substrates were labeled at the 5' end with :'lP in a reaction
mixture containing 50 mM Tris-HC1 (pH 7.5), 10 mMMgC12, 13 mM
dithiothreitol, 1p~ [-p3'P]ATP, 20 units of T4 polynucleotide kinase,
and varying amounts of oligonucleotide substrate. The reaction was
incubated at 37 "C for 80 min and was terminated by heating to
100 "C for 5 min. Oligonucleotide substrates were annealed to template DNA either a t a 1.5:l molar ratio (M13 template in excess) or
a t a 1:1 molar ratio (oligonucleotidetemplate). Annealing was carried
out by adding NaCl to a final concentration of 50 mM, then heating
the oligonucleotide template mixture to 85 "Cfor 10 min, followed by
slow cooling to room temperature over 2-4 h.
ExonucleaseAssays-For
standard exonuclease assays, reaction
mixtures (10 pl for epsilon and 15 pl for core) contained 40 mM TrisHCI (pH 7.5), 10 mM MgC12,5 mM dithiothreitol, 50 pg/ml acetylated
bovine serum albumin, and labeled duplex DNA substrate at appropriate concentrations (40 nM for c assays and 10 nM for core assays).
Reactions were initiated by adding enzyme to a final concentration
of 150 nM for or 4 nM for core. Assays were carried out a t 37 "c
(except temperature-effect assays which were carried out at 26, 30,
and 37 "C). A new preparation of pol 111 core with a higher specific
activity was used for the temperature-effect assays. At appropriate
time points,aliquots were removed and quenched in formamide/
EDTA. Samples were heated to 100 "C for 5 min, and oligonucleotides
differing in length were separated by electrophoresis in 8%polyacrylamide gels. Densitometry was carried out essentially as described
(18).For measurements of kinetic parametersof exonuclease activity,
reactions (10 pl) contained 40 mM Tris-HC1 (pH 7.5), 10 mMMgC12,
5 mM dithiothreitol, 50 pg/ml acetylated bovine serum albumin,2 nM
core, and varying amounts of substrate ranging from 25 to 850 nM.
of a correctly paired base. Autoradiographic data are shown
in Fig. 1 for the exonuclease activity of t on correct and
incorrect base pairs opposite template C. Removal of a 3'terminal G from a G. C pair proceeded more slowly than for
any of the othermispaired bases opposite C. After 8 min, less
than 10% of the correctly paired G was removed, whereas
more than 90% of the three incorrectly paired bases were
excised. The autoradiographs were scanned by a densitometer,
and thepeaks corresponding to primer bands were integrated.
The ratio of the intensity of the primer band to the sum of
the intensities of all bands present was plotted against time
using a semi-log scale. By fitting the data to a first order
exponential curve, kinetic rate constants for the exonuclease
reaction were determined. Examples of the graphed data are
shown in Fig. 2 for correct and incorrect base pairs opposite
template C (Fig. 2, A and B ) and T (Fig. 2, C and D);data
are presented for e (Fig. 2, A and C ) and for pol I11 core (Fig.
2, B and D).The rate constants are presented in Table I for
all 16 combinations for e and pol I11 core.
Both e and core preferentially attack a mispaired 3' terminus, although the relative specificities vary. We observed the
greatest specificity for mispairs opposite template C and G,
presumably because the correctly paired 3' terminus is so
resistant to exonucleolytic attack. Conversely, we found the
least specificity for mispairs opposite template A and T, for
which the correctly paired 3' terminus was relatively sensitive
to degradation. The major conclusion of our data on exonuclease specificity is that t has intrinsic discrimination for a
mispaired 3' terminus. Moreover, the properties of the e
exonuclease are qualitatively very similar to those of the pol
I11 core.
Evidence for a Melting Mechanism for t and pol 111 CoreAs noted in the introduction, there are two general mechanisms for exonucleolytic specificity toward a mispaired 3'
terminus: geometric recognition and melting capacity. For the
melting model, there are three clear predictions: (i) and A . T
correct pair should be more sensitive to exonuclease than a
G C pair; (ii) the exonuclease activity on duplex DNA should
be strongly temperature-dependent; (iii) the exonuclease activity should be most effective with a single-strand DNA
3'
/N
5' 32PJ
C
G opposite C
A opposite C
Time (min)
Primer
a
-
I
RESULTS
Exonuclease Specificity of e and pol111 Core-To determine
the specificity of pol I11 core and the isolated e subunit, we
have used a gel electrophoresis assay to measure the rate of
exonuclease action. DNA substrates were oligonucleotides,
labeled with 'lP at the 5' end, with a 3' terminus that was
either mispaired or correctly paired to theM13 template. The
velocity of exonuclease action was measured by electrophoresis of the oligonucleotide on a sequencing gel that resolves
as discrete bands single nucleotide differences in length. Assays were carried out with oligonucleotide substrates that
provided all 16 possiblecombinations of correct and incorrect
terminal pairs. The experiments were done under conditions
in which the relative velocity of the exonuclease reactions
should be proportional to the V,,,.,/K,,, specificity parameter
(substrate concentration very much less than Km).
In every case, exonucleolytic attack on an
incorrectly paired
base at the 3' terminus proceeded more rapidly than removal
opposite C
C opposite C
'
I
'
Time (min)
Primer
n
I
2
4
8
0
1
2
4
8
-
FIG. 1. Exonuclease activity o f f on mispairs opposite template C. The substrate DNA used for the assay is shown a t the top
of figure. The 20-mer oligonucleotides, each containing a different
3"terminal base, were labeled with :'2P at the5' end andannealed to
single-strand M13 DNA. Exonuclease assays were performed with
isolated c as described under "Experimental Procedures." Aliquots
from the reaction were removed at theindicated times and subjected
to gel electrophoresis. The top band in each lane represents the intact
oligonucleotide. The lower bands represent oligonucleotide substrates
that have been degraded by one and two nucleotides, respectively.
Data are shown for exonuclease assays carried out using substrates
containing 3"terminal G .C, A . C, T. C, and C.C pairs.
7890
Editing Specificity of E. coli DNA Pol 111
A
B
FIG. 2. Specificity of c and core
exonuclease for mispaired terminal
bases. A and B, activity of e and pol I11
core, respectively, on substrates containing mispairs opposite template C. 3'O), A.C
terminal bases are: G.C (-,
(---, B), T . C (--,
0), andC.C
(- -, 0).C and D,activity of e and pol
I11 core, respectively, on 3'-terminal
mispairs opposite template A. 3"termiO), G .T
nal bases are: A.T (-,
(-- -, B), T.T (- -, 01, and C.T
(- -, 0).
0
4
2
6
8
0
2
4
6
8
6
8
Time (min)
Time (min)
C
D
0
2
4
Time (min)
TABLE
I
Rate constants for exonuclease of p o l III core and isolated
6
8
0
2
4
Time (min)
effects, we carried out exonuclease assays at 26,30, and 37 "C.
The exonuclease activity withduplex D N A was compared
First order rate constants were obtained from an exponential least with that found forthe unpaired single-strand oligonucleotide.
squares fit to a plot of substrate remaining versus time, using the Examples of the data are presented in Fig. 3 for assays with
equation [SI, = (SI,* e"'eXd) (see F'g.
1 2). The kn/kco,,column gives the
pol I11 core with A opposite template T (Fig. 3A) and C
kexonumbers normalized to thecorrect Watson-Crick pair.
opposite template G (Fig. 3B). There is a notable increase in
exonuclease activity with increased temperature. A more complete set of data iscollected in Table 11. There isa very large
S"
S"
thermal effect on exonuclease for both c and pol 111 core for
c
5.3
1.0
3.4
1.0
correctly paired G. C and A . T and for the G .T mispair. There
G
36
33
6.2
11
is very high exonucleaseactivity with the single-strand D N A
N*G
A
6.1
98
19
21
substrate butonly a small thermal effect.
T
8.414
29
76
The data presented in Table I1 demonstrate that theprinciple predictions of the melting mechanism are fulfilled.
T
34
1.0
15
1.o
G
53
1.6
31
2.1
Therefore, we conclude that the melting capacity of the 3'
N-A
A
8.8
49
1.4
130
terminus is the primary recognition determinant for the ed53
1.6
130
8.3
C
iting exonuclease.
Steady-state Kinetic Parameters of ExonucleaseActivity:
A
20
1.0
4.2
1.0
V,, discrimination-To assess the relative contribution of
3.5 G
14
57
2.9
N*T
16
3.9
7.4
K,,, and V,,, to editing specificity, we carried out a steady140
20
3.3
C
65
4.7
state kinetic analysis of pol I11 core. A similar analysis was
not possible for E alone because the K, value was too high,as
G
2.3
1.0
1.9
1 .o
noted previously (12). A 35-mer oligonucleotidetemplate was
A
49
21
57
30
N*C
used to obtain substrate concentrations higher than those
T
49
20
61
31
18
23
11
obtainable with M13. The 35-mer was annealed to oligonucleC
46
otides generating a correct 3"terminal C.G pair and an
substrate. For a geometric model, A . T and G .C should be incorrect T. G pair. Conditions were determined that satisfied
equivalent, and an especially strong temperature effect would Michaelis-Menten kinetics: reaction velocity wasdirectly proportional to enzyme concentration, and reaction velocity was
not be anticipated.
constant
over the time periodmeasured (4 minfor C .G
The datapresented in Table I demonstrate a preference for
3' termini in A . T pairs over G . C; this preference is especially substrate and2 min for T. G substrate).
Reaction velocities were measured at substrate concentrapronounced whenc is actingby itself.To examine temperature
t
7891
Editing Specificity of E. coli DNA Pol III
A
l5
t
0
200
400
tsl (nu)
0
2
4
6
8
FIG. 4. Exonuclease velocity as a function of substrate concentration. Velocities were measured a t various substrate concentrations using substrates with a 3"terminal correct C. G pair (0)or
a 3'-terminal incorrect T. G pair (0).
Time (mln)
B
TABLEI11
Kinetic parameters of pol III core exonuclease
The exonuclease velocity wasmeasured a t substrate concentrations
ranging from 25 to 850 nM on substrates with either C .G or T . G
terminal pairs. Vmaxand K,,, were obtained from a linear least squares
fit to an Eadie-Hofstee plot (V/[S] uersus l/[S]).
Primer
terminus
0
e
C.G
T.G
2
0
4
6
FIG. 3. Effect of temperature on pol 111 core exonuclease.
Exonuclease assays were carried out at 26 "C (D), 30 "C (m), and
37 "C (0).A, substrate containing 3'-terminal G.C pair. B , substrate
containing 3"terminal A . T pair.
TABLE
I1
Temperature effect on exonuclease ofpol III core and c
A. Temperature effect on rate constantsfor exonuclease of pol
111 core and c"
k.. X 10' for core
37 "C
26°C
30°C
kx,,
X 10' for t
26°C
30°C
37°C
S"
S"
2.1
1.4
10
3.0
1.2
G.C
3.6
11
A.T
2.5 5.09.2
40
27
31
G.T
14
65
16
54
33
410 220210
250
Single-stranded 340280
B. Relative kexodata normalized to sinfle-strand velocity
a t each temperature
Primer 3'
terminus
Pol I11 core
e
subunit
37°C
30°C
26°C
37°C
30°C
26°C
1
1.6
2.0
2.1
4.8
1
2.1
4.5
2.9
11
G*T
1
1.7
1.9
3.1
2.7
a Rate constants at26, 30, and 37 "C were obtained as for Table I.
Rate constants were normalized to values a t 26 "C for each
substrate, then normalized to the velocity of single-strand DNA
degradation at each temperature.
G.C
A*T
K,
fml/s
nM
2.8 k 1.3
4.5
17
420 -+ 80
460 2 9 0
v,../K, X 10-4
S"
6.7
37
8
Time (mln)
Primer 3'
terminus
Vm*X
1
1
1
tions ranging from 25 to 850 nM. Fig. 4 shows a typical V
uersus [SI curve for exonuclease activity on a correctly or
incorrectly paired 3' terminus. Table I11 presents the values
for V,, and K, obtained from Eadie-Hofstee plots (average
of threeexperiments). Values for the specificity constant
V,,,/K,
were also obtained by taking theinverse of the slope
of a Lineweaver-Burke plot; these values were closely similar
to V,,JKm obtained from the Eadie-Hofstee plot (data not
shown). The values obtained for K,,, are nearly identical for
3'-terminal C.G and T . G pairs. A similar K, equivalence
was observed previously by Maki and Kornberg (12) with a
different substrate. The VmaX
value for degradation of a T G
mispair is about 6-fold higher than VmaXfor degradation of a
correct C .G pair. This V,, is the primary kinetic parameter
defining editing specificity.
The increased VmaXfor removal of the mismatched T. G
supports the concept that the source of editing specificity is
the greater melting capacity of a mispaired 3' terminus. If the
t subunit inpol
I11 core binds with similar affinity to a
matched and mismatched terminus, it can be readily shown
(and is intuitively clear) that V,,, will be proportional to the
fraction of melted 3' ends:
-
vmax
=
k,) kx E
(k.?
where 1
2
,and 1
2,are the rate constantsfor formation of melted
and annealed 3' ends, respectively, 1
2
, is the rate of singlestrand exonuclease activity, and E is the enzyme concentration. Since the fraction of melted 3' termini is greater for
mismatched bases, V,, values should be higher for mispairs.
If the source of editing specificity was direct binding recognition of a mispaired 3' terminus, discernible K, differences
wouldbe expected. Thus our kinetic dataare completely
consistent with a melting mechanism.
DISCUSSION
Specificity of Exonucleolytic Editing-Recent structural
work has demonstrated that exonucleolytic editing is a complex process requiring a communication between different
domains or subunits of the DNA polymerase. For pol I, the
exonuclease domain is not only separated from the polymerase
7892
Editing Specificity of E. coli DNA Pol 111
domain by some 30 A, but the exonuclease site can accept
The probable explanation for the quantitativediscrepancy
only single-strand DNA (13,15,22). Thus pol
for I, the editing between editing i n vivo and exonuclease i n vitro is that
at the polym- intrinsic exonucleasespecificity mustbeaugmented
“decision” appears to depend entirely on events
by a
erase site. Indeed, kinetic studies have indicated strongly thatcontribution from the polymerase site, most likely a kinetic
editing specificity in DNA replication is achieved mainly be amplification provided by inefficient polymerization from a
delayed DNA chain elongation froma mispaired 3‘ terminus mismatched 3’ terminus. Thisdelayed chain elongationwould
(16). This lag in polymerase action presumably allows more allow the exonucleasemore time to act at amispaired 3’
time for a mismatched terminal base to melt and slide into terminus before the next base insertion event (16, 28). As
the exonuclease site (14).
noted above for polI, this feature is the
major determinant of
For pol 111, the exonuclease active site is also presumably editing specificity(16).A
similarkinetic amplification of
spatially separated from the polymerase site, because the two editing has been noted recently
for phageT 7 DNA polymerase
biochemical activities reside on distinct subunits (8, 10, 12). (29). A modestkinetic amplificationfrom the polymerase step
T o examine the question
of intrinsic editingspecificity by the would boost the specificity of pol 111 into the range expected
exonuclease subunit, we have carried out a detailed compari- from i n vivo work. Thus pol 111 probably partitions its specison of the exonuclease activity of pol I11 core and t subunit. ficity determinants for editing between the intrinsic discrimWe havefound that t hasintrinsic specificity forany 3‘ ination of the exonuclease site and the kinetic contribution
terminus with a mispaired base. The specificity of E is quali- of the polymerase site.
tatively similar to thatof pol 111 core. However, the addition
REFERENCES
of the polymerase subunit markedly increases the activityof
1. Drake, J. W., Allen, E. F., Forsberg, S. A., Preparata, R., and
the exonuclease for all duplex substrates; this
effect probably
Greening, E. 0.(1969) Nature 221, 1128-1131
derives from the contribution of the a subunit to effective
2. Kornberg, A. (1980) DNA Replication, W. H. Freeman and Co.,
recognition of the 3’ terminus (12).
San Francisco
Mechanism of Exonucleolytic Editing-The initial experi3. Loeb, L. A., and Kunkel, T.A. (1982) Annu. Reu. Biochem. 51,
ments with polI and T4 polymerase strongly indicated
a
429-457
melting mechanismfor exonucleaseaction (19,20).The struc- 4. Echols, H., and Goodman, M. F. (1991) Annu. Reu. Biochem., in
press
tural analysisof pol I defined a surprisingly extreme formof
5. Kornberg, A. (1988) J . Biol. Chem. 2 6 3 , 1-4
melting mechanism; the exonuclease site could accept only
6. McHenry, C. S.(1988) Annu. Rev. Biochem. 57,519-550
single-strand DNA (13, 22). Our work with the editing exo7. Scheuermann, R., Tam, S., Burgers, P. M. J., Lu, C., and Echols,
nuclease of pol 111strongly supportsa melting mechanismfor
H. (1983) Proc. Natl. Acad. Sci. U. S. A. 8 0 , 7085-7089
both pol I11 core and the isolated t subunit. Thus the major
8. Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci.
difference between pol I and pol I11 appears tobe the intrinsic
U. S. A. 81, 7747-7751
9. Welch, M. W., and McHenry, C. S.(1982) J. Bacteriol. 152,351editing specificity of t itself. As noted below, this intrinsic
356
specificity is unlikely t o be sufficient to explain the complete
10. Maki, H., and Kornberg, A. (1985) J. Biol. Chem. 260, 12987contribution of editing to thefidelity of replication by pol 111.
12992
However, this feature of the exonuclease probably allows pol 11. McHenry, C. S.,and Crow, W. (1979) J. Bid. Chem. 254,1748111 to achieve the very high editing precision needed for a n
1753
enzyme dedicated to chromosomal duplication. Althoughwe 12. Maki, H., and Kornberg, A. (1987) Proc. Natl. Acad. Sci. U. S. A.
84,4389-4392
do notknow whether a melting mechanismwill be a universal
property of editing exonucleases, the idea is attractive, be- 13. Steitz, T. A., Beese, L., Freemont, P. S., Friedman, J. M., and
Sanderson. M. R. (1987)
.
. Cold SDrinp Harbor Symp.
~. Quant. Bid.
cause a melting mechanismallows a ready evolutionary tran52,465-472
sition from a free single-strand nuclease to an editing subunit 14. Joyce, C. M., Friedman, J. M., Beese, L., Freemont, P. s., and
or domain designed to excise misinserted nucleotides.
Steitz. T. A. (1988) in DNA Replication and Mutagenesis
Contribution of Editing to Replication Fidelity-An impor(Moses, R. E., and Summers, W. C.,-eds)pp. 220-226, American
Society for Microbiology, Washington, D. C.
tant biological question is whether the intrinsicspecificity of
the exonuclease is sufficient to explain the contribution of 15. Derbyshire, V., Freemont, P. S., Sanderson, M.R., Beese, L.,
Freedman, J . M., Joyce, C. M., and Steitz, T. A. (1988) Science
editing to the fidelity of DNA replication. An accurate esti2 4 0 , 199-201
mate of the editing concentration i n vivo is complicated by 16. Kuchta, R. D., Benkovic, P., and Benkovic, S.J. (1988) Biochemthe interplay of fidelity systems (4). The mostdefective muistry 27,6716-6725
17. Echols, H. (1982) Biochimie 64,571-575
tations in the dnaQ gene codingfor t conferaverylarge
increase in mutation rate, up to 104-105-fold (23, 24). How- 18. Sloane, D. L., Goodman, M. F., and Echols, H. (1988) Nucleic
Acids Res. 16, 6465-6475
ever, this number is an overestimate, because the mismatch
19. Brutlag, D., and Kornberg, A. (1972) J . Biol. Chem. 247, 241repair system becomes ineffective at high mutation rates (25,
248
26). The best currentguess forthe contributionof exonucleo- 20. Bessman, M. J., and Reha-Krantz,L. J. (1977) J . Mol. Biol. 1 1 6 ,
lytic editing in vivo isinthe
102-103 range (4,25). This
115-123
number is in the same rangeas a rough estimate obtained for 21. Messing, J. (1983) Methods Enzymol. 101, 20-78
pol 111in vitro by comparing theoverall fidelity of pol I11 with 22. Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., and Steitz, T.
A. (1985) Nature 313,762-766
base insertion data for pol I (27).
23. Degnen, G. E., and Cox, E. C. (1974) J. Bacteriol. 117,477-487
Our work on the exonuclease specificity of pol I11 core and 24. Cox, E. C., and Horner, D. L. (1982) Genetics 100,7-18
t indicates a much lower intrinsicdiscrimination
for the 25. Schaaper, R. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 8 5 , 8126editing exonuclease than the editing contribution to DNA
8130
replication inferredin vivo. Our datain Table I were obtained 26. Schaaper, R. M., and Radman, M. (1989) EMBOJ. 8,3511-3516
under conditions in which the relative rates of exonuclease 27. Fersht, A. R., Knill-Jones, J. W., and Tsui, W.-C. (1982) J . Mol.
Biol. 156, 37-51
activity (kex,,)should represent the relativespecificities for 28. Mendelman,
L. V., Petruska, J., and Goodman, M. F. (1990) J .
terminal nucleotides ( VmaX/Km).
This conclusion is supported
Biol. Chem. 265,2338-2346
by the detailed kinetic data
of Table 111. For pol I11 core, these 29. Donlin, M. J., Patel, S. S., and Johnson, K. A. (1991) Biochemistry, 30,538-546
specificity ratios range from 2- to 30-fold.
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