THE JOURNAL OF BIOLOQICAL CHBYIBTBY
Vol. 247, No. 1, Issue of January 10, pp. 241-248, 1972
         Printed in U.S.A.

Enzymatic Synthesis of Deoxyribonucleic Acid

XXXVI. A PROOFREADING FUNCTION FOR THE 3' + 5' EXONUCLEASE ACTIVITY IN DEOXYRIBO-

NUCLEIC ACID POLYMERASES*

(Received for publication, July 16, 1971)

DOUGLAS BRUTLAGS AND ARTHUR KORNBERG
From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

SUMMARY

The Escherichia coli and T4 DNA polymerases do not ex-
tend chains in which the 3'4erminal nucleotide (primer
terminus) is not paired with the template.  By using syn-
thetic double-stranded polynucleotides, the 3' -+ 5` exonu-
clease function of these polymerases was shown to be di-
rected specifically against a mispaired or unpaired primer
terminus.  Chain extension of such termini begins only after
all mispaired nucleotides have been removed and a base-
paired terminus is reached.  The latter is completely con-
served while polymerization is maintained.  These results
suggest that the function of the 3' --* 5` exonuclease activity
of DNA polymerases is to remove mispaired nucleotides
which have been incorrectly incorporated, thereby increasing
the fidelity of template copying.  The function of other E.
coli exonucleases suggested by their specificities on poly-
nucleotide substrates are trimming loose ends of DNA for
exonuclease I and enlarging nicks and gaps within helical
regions for exonuclease III.

An exonuclease activity which degrades DNA chains in the
3' to 5' direction is a component of DNA polymerase isolated
from Escherichia coli (1). The 3` -+ 5' exonuclease is also closely
associated with and appears to be part of DNA polymerases
induced in E. coli by infections with bacteriophages T2, T4, and
T5 (2-4). The specificity and possible functions of this nuclease
have been obscured by the presence in E. coli DNA polymerase
of another and even more active exonuclease activity that de-
grades chains from 5' to 3`. Recently it has become possible to
separate and physically isolate these two nuclease activities by
proteolytic cleavage of DNA polymerase (5, 6).  From the
parent polypeptide chain (mol wt 109,000), two active fragments
were isolated: the small fragment (mol wt 36,000) contains the
5' -+ 3' exonuclease activity; the large fragment (mol wt 75,000)
contains the 3' + 5' exonuclease activity as well as the polym-
erizing functions (5, 6).

* This study was supported in part by grants from the National
Institutes of Health (United States Public Health Service) and
the National Science Foundation. The previous paper in this
series is Reference 33.

# Predoctoral Fellow of the National Science Foundation.

The studies reported here on the specificity of the 3' 4 5'
exonuclease support earlier speculations (3,7,8) that this activity
may be designed to remove mispaired nucleotides which have
been incorrectly incorporated at the 3'-hydroxyl end of the
double-stranded DNA and thereby serves a corrective function.'
By the use of specifically labeled homopolymers and block
copolymers as substrates we show in this report that under
polymerising conditions a mispaired primer terminus is quan-
titatively removed by the DNA polymerase whereas a correctly
base-paired primer terminus is completely conserved.  It thus
appears that the 3' -+ 5` exonuclease may serve a proofreading
role in removing mispaired nucleotides at the growing end of a
chain and that the polymerase will not extend a chain until the
mispaired 3` terminus is removed.

The availability of these polynucleotide substrates has enabled
us also to examine the specificities of the E. coli exonucleases I
and 111. These nucleases appear to perform complementary
roles in trimming single-stranded ends of DNA chains and in
enlarging nicks and gaps within helical regions of double-stranded
DNA.

MATERIALS

Nucbotides-Unlabeled deoxyribonucleotides were purchased
from P-L Biochemicals except for dUTP which was purchased
from Calbiochem.  dITP was a gift from Dr. M. Fikus and was
prepared by deamination of dATP as described by Inman and
Baldwin (9).  [aH]dTTP and taH]dATP were purchased from
Schwarz BioResearch. iaH]dUTP was purchased from
Amersham-Searle and was purified before use by paper chro-
matography (Schleicher and Schuell, No. 589 orange ribbon)
using an isopropyl alcohol-concentrated NHIOH-water (7 : 1 :2)
solvent.  [CY-~*P]~TTP was purchased from ICN and was checked
for radiochemical purity by paper electrophoresis (20 mu sodium
citrate buffer, pH 3.5).  ["CIdTTP was purchased from New
England Nuclear. All specific activities were determined by
spectral measurement and radioactivity measured on Whatman
GF/C glass filters in a Nuclear Chicago scintillation counter.
2',3'-Dideoxythymidine triphosphate and the 6`-deoxy-6'-
homothymidine pyrophosphoryl phosphonate were gifts from
Dr. J. G. Moffatt.  8,y-dTTP methylene diphosphonate was
purchased from Miles Laboratories.

Enzymes--Micrococcal nuclease and spleen phosphodiesterase
were purchased from Worthington Biochemicals. Alkaline
L. E. Orgel and F. H. C. Crick, personal communication.

241

242

Enzymatic Synthesis of DNA. XXXVI

Vol. 247, No. 1

phosphatase was isolated and purified according to the method
of Malamy and Horecker (10).  DNA polymerases induced by
T4 umN82 and T4 umN82 tsL56, the gifts of Dr. W. M. Huang,
had been purified as described (3, 11).  E. coli DNA polymerase
was purified as described (12).  The large fragment obtained by
proteolytic cleavage of this enzyme appeared in the purification
procedure of the native DNA polymerase and was also purified
to homogeneity (6).  All DNA polymerases used in this study
yielded a single protein band on sodium dodecyl sulfate-polya-
crylamide gel electrophoresis.  E. coli exonuclease I was purified
through the hydroxylapatite stage (specific activity of 15,000
units per mg) (13).  E. coli exonuclease I11 had a specific activity
of 130,000 units per mg (12).  Terminal deoxynucleotidyltrans-
ferase (terminal transferase) purified from calf thymus according
to Kat0 et al. (14) was a gift from Dr. F. N. Hayes (Los Alamos
Scientific Laboratory, Ins Alamos, New Mexico).

METHODS

Polynucleotides-The polynucleotides d(T)260 and d(I)~s were
prepared with terminal transferase as described (15) except that
20 rnM potassium phosphate buffer (pH 7.0), 20 mM potassium
cacodylate buffer (pH 7.0), and 8 mM MgClz were used for the
polymerization of dITP.  d(A)mo was prepared according to
Riley, Maling, and Chamberlin (16) and d(C)looo according to
Chamberlin and Patterson (17).

Terminally Labeled Polynucleotides-Polynucleotides, radio-
activity labeled at their 3'4ermini with various nucleotides

d(T)z60-[SH]d(U) 1.8, and d(1)~,-[~H]d(T)   were synthesized by
incubating labeled triphosphates with unlabeled polynucleotides
and terminal transferase.  To add a purine nucleotide, dATP
for example, to d(T)Z60, 0.3 mu d(T)26Oa was incubated with 5 /.LN
(3HIdATP (17.7 Ci per mmole), and 0.15 mg per ml of terminal
transferase in 20 mN potassium phosphate buffer (pH 7.0), 20
mM potassium cacodylate buffer (pH 7.0), 8 mM MgC12, and 1
mM P-mercaptoethanol at 37' for an amount of time (predeter-
mined by a small scale reaction) sufficient to add about 1 mole of
nucleotide per mole of polynucleotide chain.  Incorporation was
monitored by adsorption to DEAE-paper as described below.
To add a pyrimidine nucleotide, 0.1 M potassium phosphate
buffer (pH 7.0), 0.1 M potassium cacodylate buffer (pH 7.0), and
0.25 mM CoC12 were used.  The reaction was terminated by
cooling and adding 50% KOH to a final concentration of 0.3 M.
After 10 min at O', the mixture was neutralized with 85% Hap04
and dialyzed extensively against 1 M NaCl, 10 mM Tris butrer
(pH S.O), and then uerm 10 mM Tris buffer (pH 8.0) to remove
the salt.  The final average number of residues added was
determined from the specific activity of the added nucleotide,
the length of the polynucleotide, and the radioactivity present
in the labeled polymer per mole of total nucleotide as determined
by ultraviolet absorption.

Determination of Distribution of Labeled Residues-Hayes et al.
(18) have shown that limited addition of nucleotides to short
oligonucleotide primers by terminal transferase gives a Poisson

* Polynucleotides labeled at the 3' terminus are abbreviated
as d(T)260-[aH]d(C)l,~, where 260 gives the average number of
residues in the polymer of unlabeled thymidylate to which has
been added on the average 1.1 moles of aH-labeled deoxycytidylate
per mole of polymer.
8 Polynucleotide concentrations are expressed in terms of moles
of total nucleotide.

(d(T)260-[~H]d(T)o.s, d(T)tso-['Hld(C)~~J ~(T)~BO-[*HI~(A)O.Q,

distribution of added residues.  This conclusion was found to
apply also to the longer polynucleotide primers used in this study.
This determination was made from an independent measure of
the distribution of nucleotides as well CM the average number of
residues added per chain.  Degradation of the labeled polymers
by micrococcal nuclease and spleen phosphodiesterase converts
all the internal nucleotides to 3'-nucleotides and the 3'-terminal
residue to a nucleoside.  The ratio of label converted to nu-
cleoside to the total label found in the nucleoside and 3'-
nucleotide, combined, gives the fraction of the labeled residues
at the 3`-terminal position (FJ.  For this analysis, 3 nmoles of
each of the terminally labeled polynucleotides were degraded
with micrococcal nuclease and spleen phosphodiesterase and the
products were separated by chromatography as described by
Wu and Kaiser (19).  The hydrolysis converted >98% of the
total label to nucleoside and nucleotide.  Table I summarizes
the average number of labeled residues and the fraction of these
residues which was terminal.  The observed fraction of terminal
residues was in good agreement with that predicted by a Poisson
distribution.  It can be demonstrated that this analysis is
formally equivalent to showing that the fraction of chains to
which no labeled residues were added conforms to the Poisson
distribution.

Determination of Polynucleotide Length-The number-average
length of a polynucleotide was determined by end group labeling
analysis as described by Weiss, Live, and Richardson (20).
Nuclease Assays-Nuclease assays were performed in 0.2 rnl
containing 2 nmoles of [aH]d(T)~~O, with or without 5 nmoles of
unlabeled d(A) 4000, 0.05 M N-hydroxyethylpiperazine-Wethane-
sulfonate buffer (pH 7.4), and 5 m~ MgC12.  The mixture was
was heated to the appropriate temperature and a 20.~1 sample
was taken and applied to a 1.5-cm square of Whatman DE-81
paper.  Then 10 J of enzyme diluted in bovine serum albumin
(1 mg per ml), 0.05 M HEPES4 buffer (pH 7.4), and containing
less than 1 pmole of enzyme were added to the reaction; 20.~1
samples were removed at appropriate intervals and adsorbed onto
1.5 ern squares of DE-81 paper.  The squares were washed three
times by gentle agitation for 5 min in 100 ml of ammonium
formate, 0.3 M (adjusted to pH 7.8), and dehydrated by two
washes in 95% ethanol and one in anhydrous ether.  The
squares were air dried and the amount of radioactive label re-
maining in polynucleotide determined in a scintillation counter.
(Polymerization was also measured with this technique by
using labeled deoxyribonucleoside triphosphates.) Nuclease
rates were determined from a least squares fit to initial linear
points of nucleotide hydrolyzed plotted against time.  Rates
were determined from only those assays in which >20% of the
polynucleotide had been degraded and in which the rate was
linear for at least 3 time points.

RESULTS

S' + 6` Exonuclease Rates of DNA Polymerases: Influence of
Secondary Structure and Temperature-The nuclease rate of the
large fragment (a proteolytic fragment of the E. coli enzyme
containing only polymerase and 3` -+ 5' exonuclease activities)
on d(T)300 was 4fold greater with the polymer in a

4 The abbreviations used are: HEPES, N-2-hydroxyethyl-
piperazine-N'-2-ethanesulfonic acid; ddTl'P, 2',3'-dideoxythy-
midine triphosphate; dTMPPCP, B,-pdTTP methylene diphos-
phonate.

Issue of January 10, 1972

D. Brutlag and A. Kornberg

243

I I I

a NodTTP

                TABLE I
Distribution of terminal residues on synthetic polynucleotides

I I I

b No dTTP 1

Polynucleotide composition

d(T)zso-['H]d(T) o .86

d(T) zao-['HId(C)1.11
d(T)~aa-[~Hld(A)0.8T
d(T) zao-['HId (U)I .81
d(I)zes-['IId(T)~ .6t

6 The fraction of labeled residues which are at the 3'-OH termi-
nal position of a chain (F,) was determined by nuclease degrada-
tion.  Specific activities of the labeled nucleotides were from 7
to 20 Ci per mole.

b Ft = (1 - e-r)/r, for a Poisson distribution of labeled residues
where r is the average number of labeled residues per chain.

Fraction of labeled residues at the
      3' terminus

Observeda Poissonb

______._

0.54         0.67
0.56        0.60

0.50         0.46
0.47   1 0.49

0.42 0.67

                TABLE I1
Injuence of temperature on 9' -+ 6' exonuclease rates of DNA
polymerase acting on single- and double-stranded polynucleotides

1 Substrate

I

   19
  1100
' 850
  790

1 I

4.8

350
27
11

DNA polymerase

Double-
stranded

Temperature

E. coli large fragment
T4 wild typeo
T4 wild type"
T4 L56"

37"
37
30
30

ap-Mercaptoethanol (10 mM) was included in assays of the T4

enzyme.

single-stranded as compared to a double-stranded conformation
(d(T) $O@:d(A) 4000) (Table 11).  A similar preference for single-
stranded d(T)sao was observed at 37" with the T4 enzyme which
has a much higher nuclease activity than the E. coli enzyme.

When the nuclease rates of the T4 polymerase on single-
stranded d(T)3oo at 30" are compared with the rates at 37" only
a slight reduction in rate (23%) was found.  However, there was
a 13-fold reduction in rate on the double-stranded substrate
(Table 11).  A similar marked temperature dependence of the
nuclease rate of the E. coli large fragment on double-stranded
substrates has also been observed.6  This temperature depend-
ence indicates a high energy of activation for some rate-limiting
step on double-stranded substrates, possibly local denaturation
at the 3' terminus.  Thus the 3' 3 5' exonuclease might require
that the terminal nucleotides not be base-paired (i.e. frayed).

Nucbase Action of DNA Polymerases on Primer Terminus-
Polynucleotides containing on the average about one radioactive
nucleotide residue per chain at the 3' terminus were synthesized
employing terminal transferase (Table I).  The terminal residue
was either the same as the rest of the polymer or distinctive from
it.  The distribution of labeled residues added to the polynucle-
otide chains approximates a Poisson distribution.  For simplic-
ity, these 3' terminally labeled polymers are referred to with a

6 P. Setlow and A. Kornberg, unpublished results.

FIG. 1. Specificity of the 3'- 5' exonuclease action of the large
fragment of the Escherichia coli DNA polymerase on terminally
labeled polynucleotides.  An excess of large fragment (5.4 pmoles)
was incubated at 37" under standard assay conditions with the
indicated polymers and triphosphates.  Samples were taken at
indicated times.  a, 0.6 nmoles of d(T)~,o-['H]d(T)l with and
without 5.4 nmoles of d(A)roao; b, 0.66 nmole of ~(T)~so-[~H]~(C)I
with and without 5.4 nmoles of d(A),000; c, same as a with d(A)raoo
and with 50 p~ [dZP]dTTP(18,000 cpm per nmole) ; d, same   b
with d(A)tooo and with 50 pd [o(-32P]dTTP.

subscript "1" even though the actual average number of labeled
residues only approximates unity.
The rate of removal of the terminal label from ~(T)PBo-[~H]~(T)~
by the large fragment was too great to permit accurate rate
measurements (Fig. la).  However, the decrease innuclease rate
with the polynucleotide in a double-stranded conformation was
large enough to be apparent.  Thus the rate of hydrolysis of
these terminal residues reflects the preference for single-stranded
substrate initially observed with uniformly labeled  polynu-
cleotides. The terminal residue of d(T)2eo-[WJd(C) I, which
does not base pair with d(A)4ooa, was removed at a rapid rate
both in the single and the double-stranded conformation (Fig.
lb).  Although the relative rates of hydrolysis of dCMP in these
two cases cannot be determined, the rate of removal of dCMP
from d(T)260-[3H]d(C)1:d(A)rooo was far more rapid than that of
dTMP from d(T)zso-[8Hld(T)1:d(A),rm (compare Fig. 1, a and b).
Thus the preference of the nuclease for nucleotides which are not
able to base pair with the template is readily apparent.

Removal of Terminal Nucleotides Prior to Polymerization-
Polymerization had a dramatic effect on the 3' ---f 5' exonuclease
activity.  When the large fragment and the polymer d(T)PBO-
['H]d(T), :d(A)dOOO were incubated with dTTP so that polymeri-
zation could proceed, there was no detectable loss of terminal la-

Vol. 247, No. 1

Enzymatic Synthesis of DNA. XXXVI

FIQ. 2. 3' + 5' exonuclease action of the large fragment on the
terminal residues of d (T)lao-[PzP]d(T)s-[aH]d(C) 16' d(A)4000.  The
large fragment (5.4 pmoles) was incubated under standard as-
say conditions at 37" with the following; a, 0.75 nmole of d(T)2so-
[a2P]d(T)I-[aH]d(C)la and 2.0 nmoles of d(A)mo; b, same &s a but
with 50 p~["c]d'M'P (10,400 cpm per nmole).

bel during the incorporation of d"TP (Fig. IC).  The incorpora-
tion of dTTP completely protected the terminal nucleotides from
exonuclease action.  In a similar experiment in which d(T)ZBO-
[aH]d(T)l was present in a single-stranded conformation, there
was no detectable incorporation of dTTP nor did the dTTP
protect the terminal nucleotides from nuclease.  Thus both the
template and the triphosphate which participate in po-
lymerization are required for protection of the terminal residues.

A third requirement for the protection of a terminal residue
from exonuclease action is that it be base-paired to the template.
When d(T)~ao-[~H]d(C)~:d(A)400o was incubated with polym-
erase and dTTP, the terminal dCMP residue was removed
rapidly after which polymerization proceeded normally with the
primer that remained (Fig. Id).  After extensive polymerization
little or no [aH]dCMP was detectable in the product, indicating
that the DNA polymerase does not polymerize on a mispaired
3' terminus; instead the enzyme first removes the non-base-
paired nucleotides by 3' + 5' exonuclease action.

In order to investigate further how strict is the base pairing
specificity of this exonuclease during polymerization, a copolymer
was synthesized containiing a block of 5 [c+P]dTMP residues
subterminal to a block of 16 PHIdCMP residues (d(T)~o-[~P]d-
Qs-[aH]d(C)~~:d(A),o~o).  The fate of the terminal block of 16
non-base-paired dC residues and the subterminal block of base-
paired dT residues were both observed.  In the absence of dTTP,
the enzyme (large fragment) removed the terminal dCMP resi-
dues at a maximum rate of 20 nucleotides per min per chain,
whereas the dTMP residues were removed only after a significant
lag and then only at a maximum rate of 4 residues per min per
chain (Fig. 2u).  The rate difference is not immediately appar-
ent from Fig. 2a because the plot of the percentage of residues
remaining does not reflect that there are initially three times as
many dCMP residues as dTMP residues.  When dTTP was
included so that polymerization could proceed, the terminal
dCMP residues were still removed as rapidly but the subterminal,
base-paired dTMP residues were retained (Fig. 2b); loss of dTMP
was less than 0.5 residue per chain. As expected, the
polynucleotide supported synthesis only after a lag, during which
the dCMP residues were removed.  This experiment indicates

               TABLE I11
Inhibition oj 3' -+ 5' exonuclease is specific for deozyribonucleoside
      triphosphates which are incorporated

The deoxyribonucleoside triphosphate, 50 pra, was incubated
with 0.6 nmole of d(T),,o-[*Hld(T)~, 2.0 nmoles of d(A)tooo, and
5.4 pmoles of large fragment at 37" under standard assay condi-
tions for 4 min. Incorporation was followed using 14C- or **P-
labeled triphosphate (10,OOO cpm per nmole) except where noted.

I I

TriphosphFte Labeled primer

terminus

incorporationD remainin2

Nucleoside triphosphate added

dATP
dGTP
dCTP
Homophosphonate'
dTMPPCP
dTTP
dUTP
ddTTP

None
None
None
Noned
Noned
Normal
Normal
One

% o/ initial
  <5
  <5
  <5
<5
<5
100

95
97

6 "None" indicates no incorporation detectable (less than resi-
due per chain); "nor nal" indicates rapid chain extension; "one"
indicates a single residue incorporated per chain (22).

Values of <5% indicate that both the rate and the extent of
hydrolysis were the same as a control without any deoxyribonu-
cleoside triphosphate added.

c 6'-Deoxy-6'-homothymidine pyrophosphoryl phosphonate.
d The inability of DNA polymerase to incorporate these ana-
logues was shown by their inability to replace dTTP in a poly-

[d(A-T)] primed reaction (Brutlag and Kornberg, unpublished
results).

nucleotide, but also the inability of the polymerase to utilize
such a nucleotide as a primer terminus for polymerization.
Inhibitioa of S' -+ 6' Exonuclease by Deoxyribonucleoside
Triphosphates-The previous expcrimcnts demonstrated that
dTTP in an exonuclease assay with the base-paired template
primer d(T)~~~-[~wd(T) :d(A)tooo completely inhibited the 3' -+
5' exonuclease activity of the DNA polymerase on terminal
dTMP residues. Incorporation of nucleotides on a terminally
labeled polynucleotide covers the labeled residue and protects it
from exonuclease action.  In order to determine whether incor-
poration of the triphosphate is required for protection of a ter-
minal nucleotide, other deoxyribonucleoside triphosphates were
tested for their ability to inhibit exonuclease (Table 111).  Nu-
cleotides not complementary to the template d(A)cooo (i.e. dATP,
dGTP, and dCTP) were not incorporated and also failed to in-
hibit exonuclease. The phosphonates, dTMPPCP and 6'-
deoxy-6'-homothymidine pyrophosphoryl phosphonate, are
analogua of dTTP, which bind to the triphosphate-binding site
on the enzyme (21),6 and have the same base pairing specificity
as dTTP. Neither analogue is incorporated by DNA polym-
erase nor does either analogue inhibit exonuclease.  Three nu-
cleotides which were incorporated (dTTP, dUTP, and ddTTP)
all inhibited exonuclease (Table 111).

                              Since
ddTTP lacks a 3'-hydroxyl group only 1 residue is incorporated
per chain (22). Nevertheless, this limited incorporation was
sdcient to protect essentially all of the terminal nucleotides
from exonuclease. Secondly, ddTTP is incorporated at a rate

The behavior of ddTTP is of interest in two respects.

not only the specscity of the 3' -+ 5' exonuclease for amispaired

8 D. Brutlag and A. Kornberg, unpublished results.

Issue of January 10, 1972

D. Brutlag and A. Kornberg

245

E. coli large fragment.. ..
T4 wild type. ...........
T4 W6.. ................

                 TABLE I\'
Yispairing of 3'-terminal nucleotide as basis of nuclease action by
      large fragment and TQ DNA polymerase

An excess of DNA polymerase (4 to 5 pmoles) was incubated
with 0.6 nmole of primer polynucleotide d(T)m (first three col-
umns) or d(I)%Ba (fourth column) labeled at the 3' terminus with
the designated (*) nucleotide and annealed to 5 pmoles of a tem-
plate (d(A)tooo or d(C)lm).  dTTP or dGTP (50 p~) was present,
as indicated.  Incubation was 4 min under standard assay condi-
tions except that 10 mM p-mercaptoethanol was included in assays
of the T4 polymerase.  The temperature was 37" except as noted.

yo of indial labeled primer terminus remaining

98 3 2 3
97
100"

--&OH  --TT<oa  --'rT~oa  --I I'To B:
--AAAAA-  --AAAAA--  -AAAAA-- --CCCCC--

DNA polymerase

1 d?TP 1 dTP I dTTP I dGTP

loo

53
100

100

<0.5

47
<0.5

<0.5

a These assays were performed at 30".

which is a 1000-fold less than that of dTTP (22).  Although
this rate of ddTTP incorporation (0.4 nucleotide per chain per
min) is 10-fold slower than that of exonuclease action on the
terminal base-paired residues in the absence of a triphosphate,
the presence of ddTTP completely inhibited the exonuclease.
Thus the ability of a nucleoside triphosphate to bind to the
triphosphate binding site on the enzyme and to base pair with
the template strand is not sufficient to inhibit exonuclease.  It
is required that the triphosphate establish and maintain an
effective polymerization complex with the template and primer
terminus, even if the completion of the phosphodiester bond is
exceedingly slow.

Comparison of SpeciJcity of E. coli and T4 DNA Polymerases
in 3' ---t 5' Hydrolysis of Various Mismatched Primer Termini-
The T4 polymerase showed the same capacity as the E. coli
enzyme to preserve the terminal dTMP residue of d(T)m
I3H]d(T)1 :d(A),ooo during polymerization and to remove the
terminal dCMP residue of d(T),so-[*H]d(C) 1:d(A)4000 (Table IV).
These enzymes also effectively removed a purine nucleotide from
a purine-purine mismatch (d(T)zao-[aH]d(A) l:d(A)4000) and a
pyrimidine nucleotide from a pyrimidine-pyrimidine mismatch
(d(I)za-[aH]d(T)I :d(C)looo).  With the latter polymer incubated
in the presence of dGTP, there was efficient removal of the ter-
minal dTMP residue (Table IV).  This primer then supported
incorporation of dGTP. The polymer d(T)2~-['H]d(U)1:
d(A)4ooo, in which the terminal dUMP residue base pairs with the
template, was also tested with the E. coli large fragment.  The
result, as expected, was that more than 96% of the dUMP residue
was retained in the presence of either dUTP or dTTP.

It is of interest to compare the base pairing specificity of the
3' - 5' exonuclease activity of the mutant T4-induced DNA
polymerase (L56) with that of the wild type enzyme.  A muta-
tor role has been proposed for thc cnryme in T4 L56, a tempera-
ture-sensitive mutant in gene 43, the gene which codes for the
DNA polymerase (23, 24).  The exonucleases of both T4 DNA
polymerases had approximately the same rate at 30" (Table I)
and both showed base pairing specificity identical with the E.
coli large fragment (Table IV).  Terminal base-paired residues
were retained during polymerization whereas mispaired residues
were removed.  The base pairing specificity of the mutator

             TABLE V
Nature of cleavage of terminally labeled polynucleotides
   in presence of inorganic pyrophosphate

E. coli large fragment (7.5 pmoles) was incubated with 1 mM
pyrophosphate, 1.2 nmoles of d(T)~60-[aH]d(T)1 or 1.4 nmoles of
d(T)?e0-[~H]d(C)1, with or without 2.1 nmoles of d(A)4000 under
standard assay conditions at 37" for 6 min.  The products were
adsorbed to Norit, eluted, and analyzed by paper electrophoresis
(20 mM sodum citrate buffer, pH 3.5).  The fraction of the radio-
activity migrating as monophosphate ((JH]dTMP or ['HI-
dCMP and as triphosphate [('HJdTTP or (rH)dCTP) was deter-
mined.

Structure at 3' primer terminus

Single-stranded chain (d(T)zea-['H]d(T)1) ...

d(Ti):d(A)tooo). ..........................
Single-stranded chain (d(T),60-[~H]d(C)i). ...

Double-stranded, base-paired (d(T)n60-['H]-

Double-stranded, mispaired (d(T)z60-['H]-

d (C)i:d(A)tooo). ..........................

Tim4 in minutas

FIG. 3. Exonuclease I action on single strands and on base-
paired and mispaired 3' termini.  Escherichia coli exonuclease I
(1.4 units) was incubated under standard assay conditions at 37'
with 10 mM p-mercaptoethanol and the following polynucleotides:
a, 0.6 nmole of d(T)~ao-[*Hld(T)~ with and without 2.7 nmoles of
d(A)tooo, b, 0.66 nmole of ~(T)z~o-[~H]~(C)I with and without 2.7
nmoles of d (A)~ooo and 0.54 nmole of d (T)zso-[*H]d (C) 1) annealed
to 2.7 nmoles of d(A)tooo.

DNA polymerase appears at this level of analysis not to be
markedly different from the wild type enzyme.  However, a 1 %
failure by the mutant polymerase to remove a mispaired primer
terminus would not have been detected above the background in
our assay.

Pyrophosphorolysis Is Reversal of Polymerization and Is Dia-
tinct from 3' -+ 6' Exonuclease-Suggestions that the 3' --* 5'
exonuclease activity of the E. coli DNA polymerase is analogous
to pyrophosphorolysis (25, 26) appeared unlikely on the basis of
later evidence (27).  The requirement for d the components of
the polymerization reaction argued that pyrophosphorolysis is
the reversal of polymerization; the ability of the 3' -+ 5' exonu-
clease activity to degrade single-stranded DNA was taken as

246 Enzymatic Synthesis of DNA. XXXVI VOl. 247, so. 1

.-

0

E
?I
{ 50.

-

-
-

I 1 I I I1 I

__ -

evidence that this hydrolytic reaction was distinct from
pyrophosphorolysis which requires a double-stranded DNA.  The
availability of primer templates in which only the terminal
nucleotide of the primer is mispaired provides an even more
exacting test of the requirement of pyrophosphorolysis for a
base-paired 3' terminus.

When 1 mM pyrophosphate was present, the sole radioactive
products of degradation of single-stranded d(T)z60-[aH]d(T)1
and d(T)~so-[~H]d(C)1 by the large fragment were still mono-
phosphates: [aHJdTMP and [*H]dCMP, respectively (Table V).
This result verifies that 3'-terminal nucleotides of single-stranded
chains are not removed by pyrophosphorolysis.  With the
double-stranded polynucleotides d(T)~so-[~H]d(T) 1 : d(A) 4000 and
d(T)z6~-[aH]d(C)~:d(A)4~~~ as substrates, 47% of the [aH]dT
residues were released by pyrophosphorolysis but there was no
detectable pyrophosphorolysis of the non-base-paired [3H]dC
residues (Table V). These results establish that pyrophospho-
rolysis has the specificity expected for the reversal of
polymerization and that its characteristics are distinct from
those of 3' -+ 5' exonuclease action.

Base Pairing Specificities of E. coli Exonucbase I and Exonu-
clease IZI-Exonuclease I degrades double-stranded DNA at a
40,000-fold slower rate than denatured DNA (13).  The speci-
ficity of this enzyme was further analyzed with terminally
labeled polynucleotides.  With enzyme present in excess over
the available 3' termini, the terminal nucleotide was removed
rapidly from d(T)~~o-[~Hld(T)~ but only slowly (if at all) from
the same polymer annealed to d(A)rooo (Fig. 3a).  Attack on
the mispaired terminal nucleotide of d(T)~so-[~H]d(C) 1 was
similar (Fig. 3b).  Whereas, removal of the dCMP residues
from the single-strand was rapid, removal from d(T)Zt0-
[aH]d(C)l:d(A)tooo was very slow.  When the number of ter-
minal dCMP residues was increased to 16, exonuclease I re-
moved up to 60% of the dC residues of d(T)zao-[aH]d(C)Is:
d(A) doo0 rapidly, but the remaining nucleotides were removed
very slowly.  These data indicate that exonuclease I removes
unpaired nucleotides that are within 6 to 8 residues of a base-
paired region very slowly compared with nucleotides in a single-
stranded chain. Thus exonuclease I shows a general specificity
for extended single-stranded structures.

Exonuclease 111 (a 3' - 5' exonuclease which attacks native

DNA preferentially (28)) shows a specificity for double-stranded
structure. The terminal residues of d(T)260-[~H]d(T) were
hydrolyzed very rapidly by exonuclease I11 only when the pol-
ymer was annealed to d(A)4ooo (Fig. 4a).  Likewise, most of the
residues of d(T)260-[~H]d(C) 1 were sensitive to exonuclease I11
only when the polymer was in the double-stranded conformation
(Fig. 4b).  In experiments with polymers that contained blocks
of dCMP residues, resistance to hydrolysis increased in propor-
tion to the length of the sequence that did not anneal to the
d(A)tooo. From these results it appeared that exonuclease I11
hydrolyzes double-stranded polymers which contain one, two, or
even three mispaired terminal nucleotides at a rapid rate.  About
90% of the terminal residues were removed from d(T)260-
[aH]d(C)l:d(A)l~o~ in 4 min (Fig. 4b).  But polymers with 5 or
more residues were attacked very slowly, if at all, as 10% of the
terminal residues of d(T)~~o-[~H]d(C)1:d(A)~ooo remained and a
Poisson distribution predicts that 10% of the total label resides
in chains with 5 or more labeled residues.

DISCUSSION

Speci$eity of 3' -+ 5' Exonuclease Associated with DNA Polym-
e-rases-The 3' -+ 5' exonuclease activity of the E. coli and the
T4 DNA polymerases degrades both single- and double-stranded
DNA.  Temperature had no more than the usual influence on
degradation of single strands.  However, with double-stranded
DNA, the influence of temperature was profound.  An increase
of only 7" (from 30" to 37") enhances the rate more than 10-fold.
This behavior suggests that it is the frayed end of a helix which
is required for action of the nuclease, as does an alkaline pH
optimum (l), at which melting of the helix is favored.  These
suggestions, indicating a requirement of the nuclease for an
unpaired 3'-hydroxyl terminus, have been confirmed by our
studies with synthetic DNA polymers.

In the studies reported here, homopolymer pairs were used as
nuclease substrates in which the 3'-hydroxyl end of the primer
was either a proper base pair with the template or any of the
several kinds of possible mispairings.  When the triphosphates
appropriate for polymerization were present, then two things
were found. A properly base-paired terminus was extended
without measurable nuclease action, whereas a mispaired termi-
nus was quantitatively removed before polymerization was
initiated. Mispaired termini included purine-purine and
pyrimidine-pyrimidine pairs as well as purine-pyrimidine mis-
matches.

Suppression of 3' + 5' Exonuclease by Polymerization-What
components of the polymerization reaction are required to
suppress the 3' -+ 5' exonuclease?  We have found that only
deoxyribonucleoside triphosphates which match the template
and can be incorporated into polymers suppress nuclease action.
Phosphonate analogues of dTTP which bind to the triphosphate
site and are suitable for base pairing with a poly(dA) template
do not serve as substrates and are correspondingly ineffective in
suppressing nuclease.  Of special interest is the dideoxyribonu-
cleotide analogue (ddTTP) which is incorporated at a rate 1000-
fold slower than that of dTTP and to the extent of only
1 nucleotide per chain.  Nevertheless ddTTP inhibits exonu-
clease activity as completely as does dTTP.  These results
indicate that the primer terminus in its complex with template
and triphosphate is invulnerable to nuclease even though the
rate-limiting step in polymerization is exceedingly slow.

Issue of January 10, 1972

D. Brutlag and A. Kornberg

247

Pyrophosphorolysis as Reversal of Polymerization-Template-
directed chain extension as catalyzed by DNA polymerase re-
sults in a properly base-paired 3' terminus which is utilized for
subsequent polymerization.  Pyrophosphorolysis, as the rever-
sal of polymerization, would be expected to show specificity for
the product of polymerization, i.e. a base-paired 3' terminus.
The experiments described show that only such a terminus is
removed by pyrophosphorolysis.  A mispaired primer terminus
was not attacked by pyrophosphate.  If such a terminus can be
removed by pyrophosphorolysis, then it must be at a rate very
much slower than that of hydrolysis.  This specificity clearly
distinguishes pyrophosphorolysis which attacks only base-paired
termini from hydrolysis in which attack is only on unpaired
termini.  Thus there may be two forms of an enzyme-DNA
complex.  One form, with the primer terminus base-paired,
would allow incorporation of a triphosphate or attack by pyro-
phosphate, and the other form, in which the 3' terminus is frayed
would be available only for hydrolysis.

Proofreading Role for 3' + 6' Exonuclease-The observed
specificity of the 3' + 5' exonuclease during polymerization
suggests that this nuclease may act as a proofreading mecha-
nism to remove a mismatched nucleotide. Inasmuch as the
polymerase cannot extend a mispaired terminus, its exonuclease
would remove a mismatched nucleotide generated by itself as
well as those in the synthetic polymers studied here.  The
fidelity of template copying would be far greater as the result of
having two base pairing selection steps: first the selection of a
nucleotide for polymerization, and second, a determination
that the primer terminus is properly paired before adding the
next nucleotide.  The presence of the same base pairing speci-
ficity in the exonuclease activity of two different DNA polym-
erase3 (T4 and E. coli) points to the fundamental importance of
the esonuclease for accuracy in a template-directed polymeriza-
tion process.  Englund (8) has also suggested a role for the
exonuclease of the T4 DNA polymerase in fidelity of template
copying based on removal of unpaired nucleotides by this enzyme
prior to polymerization.  We have recently investigated yet a
third DNA polymerase, DNA polymerase I1 isolated from E. coli
by Kornberg and Gefter (29), and have shown that this enzyme
may also contain a 3' exonuclease activity with this same
specificity as greater than 80% of a base-paired terminus was
retained during polymerization, while a mismatched terminus
was quantitatively removed.

Deject in S' -+ 6' Exonuclease May Be d4uutagenic-Gene 43 of
T4 codes for the DNA polymerase and certain lesions in the gene
are known to have a profound effect on the spontaneous mutation
rate (24, 30, 31).  Allen et al. (32) have suggested on genetic
grounds that the exonuclease activity of the T4 polymerase is
involved in the fidelity of replication.  If the proofreading
exonuclease function of the polymerase is important for repli-
cation accuracy, then an alteration in exonuclease might cause
mutagenic effects.  A known mutagenic polymerase has been
purified, and Hall and Lehman (11) have demonstrated in an in
vitro experiment with the polymerase induced by T4 L56, a 4-
fold increase in misincorporation of dTTP with a poly(dC) tem-
plate as compared with the wild type T4 polymerase.  We tested
the L56 polymerase to determine whether it is defective in its
ability to remove a mispaired nucleotide.  In none of the cases
tested was the mutator polymerase significantly different from
the mild type polymerase in its exonuclease specificity (Table IV) .
In an experiment with polyinosinic acid, terminally labeled with

                T.~BLP; VI
Summary of action of E. coli 9' -+ 5' exonucleases and D.VA
     polymerase un various 3' termini at 97"
The structures depicted are a base-paired 3' terminus, an nn-

paired 3' terminus (mismatched), and a displaced 3'-terminal
strand containing many unpaired nucleotides.

Structure

fiIIIlllIlllIII:llll~llll~ll~

3'

Base-paired terminus

Mismatched terminus

Displaced strand

Action on 3' primer terminus

Chain
rtcnsion
IY DSA
Ply-
merase

-

Yes

No

NO

I' - 5'
ydroly.
sis by
DNA
Poly-
nerase
-

N 0'

Yes

Yes

-

I' - 5'
Iydrol-
rsis by
:xonu-
lease 1

-

NO

Nob

Yes

-

I' - S'
Iydrol-
isis b
Konucfe-
ise 111

-

Yes

Yes

NO

a In the presence of triphosphates.
* Hydrolysis is very slow.

dTMP residues and annealed to d(C)looo (the case most closely
resembling the conditions of Hall and Lehman), there was less
removal of the mispaired primer dT terminus by the mutator
polymerase. However, more rigorous and sensitive assays,
such as measuring direct transfer of dGTP to the terminus, will
have to be performed to establish differences.  Studies with
other mutator DNA polymerases, as well as more extensive
evaluation of the base pairing specificity, including all 12 possible
nucleotide mismatches, will help in providing insights into the
function of this nuclease.

Comdinated Roles of Exonuclease I and Exonuclease III-The
specificities of the DNA polymerase and the other exonucleases
of E. coli demonstrate that they are capable of coordinated
hydrolytic action on a variety of structures at the 3' terminus
(Table VI).  Displaced single-stranded 3' termini which might
result from various processes in repair, recombination, and
replication of DNA would be rapidly degraded by either exonu-
clease I or the 3' + 5' exonuclease of DNA polymerase.  Once
the length of the displaced strand is reduced to 5 or 6 nucleotides,
exonuclease I action becomes very slow, but the specificity of
exonuclease I11 (or again, the DNA polymerase) would allow
degradation into the base-paired region (Table VI). The
nuclease activity of the DNA polymerase on displaced single
strands is similar to the combined action of exonuclease I and
exonuclease 111.  In a double-stranded region of DNA, the DNA
polymerase would begin chain extension, whereas exonuclease
I11 would continue hydrolysis leaving a single-stranded region or
a gap.  Gaps might also be produced by DNA polymerases if the

amount of DNA precursors was severely limited.  Such gaps
might be important in the metabolism of DNA.  The redundant
action of these enzymes, and also their multifunctional nature
indicates that each may be involved in more than one physio-
logical process.  Their redundant nature would insure the in-
tegrity of the metabolism of DNA and their multifunctional

248

Enzymatic Synthesis of DNA. XXXVI

Vol. 247, No. 1

character would insure proper coordination of these vital mo-
lecular events.

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