THE JOURN~~ OF BIOLOGICAL CHEMI8TRY
Vol. 247, No. 1, hue of January 10, pp. 232-240, 1972
         Pn`ntcd in U.S.A.
Deoxyribonucleic Acid Polymerase: Two Distinct Enzymes
in One Polypeptide
11. A PROTEOLYTIC FRAGMENT CONTAINING THE 5` 3 3' EXONUCLEASE FUNCTION.  RESTORA-
TION OF INTACT ENZYME FUNCTIONS FROM THE TWO PROTEOLYTIC FRAGMENTS*
(Fteceived for publication, July 16, 1971)
PETER SETLOWS AND ARTWR KORNBERG
From the Department of Biochemistry, Stanford University School of Medicine, Stanford, Cdijmia 9,4305
SUMMARY
The small fragment (mol wt 36,000) produced by the lim-
ited proteolytic cleavage of DNA polymerase (mol wt 109,000)
retains only the 5' + 3' exonuclease activity.  The small
fragment resembles the 5' -+ 3' exonuclease of the intact
enzyme in degrading DNA to mono- and oligonucleotides and
in its capacity to excise mismatched regions such as thymine
dimers.  It differs from the intact enzyme in that deoxy-
ribonucleoside triphosphates, which support polymerization,
fail to stimulate the exonuclease or to increase the proportion
of oligonucleotides among the products.  However with a
mixture of small fragment, large fragment (mol wt 76,000;
polymerase and 3` .-* 5' exonuclease functions), nicked DNA,
and suitable deoxyribonucleoside triphosphates, the same
influences of polymerization on 5' -+ 3' exonucleases are
seen as with the intact enzyme.  Thus at the locus of a nick
in DNA, the two fragments bind adjacent to one another to
perform the coordinated polymerization-5' -+ 3' exonuclease
functions that characterize the intact enzyme.
Limited proteolysis of Escherichia coli DNS polymerase (mol
wt 109,000) splits this single polypeptide chain into two frag-
ments which retain the several catalytic activities of the intact
enzyme (1-6).  The large fragment (mol wt 76,000) which polym-
erizes deoxyribonucleotides (polymerase) and hydrolyzes DNA
in the 3' -+ 5' direction (3' + 5` exonuclease) (1,3) was described
in the preceding paper (6).  The smdl jragnent (mol wt 30,000)
retains only the ability to hydrolyze double-stranded DNA in
the 5' -+ 3' direction (5` -+ 3' exonuclease) (3, 7) and is the sub-
ject of this report.
Unlike the intact enzyme, the small fragment has no detectable
* 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 6.
$ National Science Foundation Postdoctoral Fellow.  Present
address, Department of Biochemistry, School of Medicine, Uni-
versity of Connecticut Health Center, Hartford Plaza, Hartford,
Connecticut 06105.
polymerase activity, and no exonuclease activity on single-
stranded DNA.  However, the products of the action of the
small fragment on double-stranded DNA are identical with
those generated by the 5' -+ 3' exonuclease of the intact enzyme
since both enzymes excise mono- and oligonucleotides and also
thymine dimers (8, 9).  Neither enzyme exhibits detectable
endonuclease activity on a variety of substrates.
The two dserent fragments can bind ne't to one another at a
diester bond break (nick) in a DNA molecule, and this adjacent
binding permits concomitant action of the polymerase and 5' +
3' exonuclease activities.  Simultaneous action of the two frag-
ments restores properties of the intact enzyme such as the stim-
ulation of 5`  3` exonuclease by concurrent DNA synthesis, and
the autocatalytically primed synthesis of poly[d(A-T)]?  How-
ever, the individual fragments show no direct affinity for one
another in either the presence or absence of DNA.
EXPERIMENTAL PROCEDURE
Materials
Nucleotides and P~lynucZeotides-[~~C]dATP and [14C]dCTP
were purchased from SchwarB BioResearch; other deoxyri-
bonucleotides were obtained as described previously (6).  The
1 The abbreviations used are: poly[d(A-T)], alternating co-
polymer of deoxyadenylate and deoxythymidylate; d(T),, d(T)*,
d(T)5, oligonucleotides (5',3') of deoxythymidylate containing
2, 3, and 5 residues, respectively, all with a 3'-hydroxyl and a
5'-phosphate terminus; d(C) 8, an oligonucleotide (5',3') of deoxy-
cytidylate of 3 residues in length with a 3'-hydroxyl and a 5'-
phosphate terminus; pppTpTpT, thymidylyl- (5',3`)-thymidylyl-
(5',3')-thymidine 5'-triphosphate; d(A)tooo, polydeoxyadenylate
of length about 4OOO residues; d(T)taoo, polydeoxythymidylate
of length about 4000 residues; d(1) 1000, polydeoxyinosinic acid of
length about 1000 residues; d(T)200, polydeoxythymidylate of
length about 200 residues; poly d(A-T)looo, double-stranded,
alternating copolymer of deoxyadenylate and deoxythymidylate
of length about loo0 residues; (e), thymine dimer; d(C)no-d(T)rW,
a block copolymer containing about 120 residues of deoxycytidyl-
ate on the 5' side of the molecule, and about 160 residues of deoxy-
thymidylate at the 3' side; d(C)tzo polydeoxycytidylate of length
about 120 residues; BSA, bovine serum albumin; PMSF, phenyl-
methylsulfonyl fluoride; $f5pTp(T)too, a 5'-triphosphate-termi-
nated d(T)too with the 0 and y phosphates labeled with [s*P]; ppp-
TpT, thymidylyl-(5', 3`)-thymidine 5'-triphosphate; SDS, sodium
dodecyl sulfate.
232
Issue of January 10, 1972
P. Setlow and A. Kornberg
233
oligonucleotides d(T)p, d(T),, d(T)I, and d(C)r were prepared
according to Weimann, Schaller, and Khorana (10) and [s, y"P]-
pppTpTpT was a gift from Dr. N. R. Cozzarelli (11).
d(A)400o and d(T)woo were prepared and purified according to
Riley, Maling, and Chamberlin (12); [14C]d(A)a~~o and [aH]-
d(T)4000 were prepared in an identical manner using [WIdATP
and [aH]dTTP. d(I)1ooo was prepared and purified according
to Chamberlin and Patterson (13).  The synthesis of [aH]d(T)~oo
and the addition of a 2',3'-dideoxythymidylate terminus to the
3' end of an oligo(dT) have been described previously (8, 14).
Ls, -Y-"PIPPPT~T)zoo was prepared from W, T-~PIPPPTPTPT
and [aH]dTTP using terminal transferase according to the method
of Cozzarelli, Kelly, and Kornberg (11). Activated calf thymus
DNA and activated poly[d(A-T)lo~o] were prepared by limited
digestion with pancreatic deoxyribonuclease (15, 16).  Concen-
trations of oligo- and polynucleotides are expressed BS nucleotide
residues.
Enzymes-E. coli DNA polymerase (Fraction VII) was used
and had a specific activity of 4500 units per mg with activated
calf thymus DNA (4). The large fragment of DNA polymerase
was prepared either as described in this communication or puri-
fied from E. coli as described previously (6).  No differences
were observed between the two preparations (6).  Pancreatic
deoxyribonuclease was purchased from Worthington Biochemi-
cals. Terminal deoxynucleotidyltransferase from calf thymus
(17) was a gift of Dr. F. N. Hayes (Los Alamos Scientific Labora-
tory, Los Alamos, New Mexico), and subtilisin (Carlsberg) (18)
was a gift of Dr. T. Link.
Methods
Polymerase and Exonuclease Assays-The assay of DNA syn-
thesis on activated calf thymus DNA has been described (6),
and the specific assays for 3' 4 5' exonuclease and 5' + 3' exo-
nuclease were carried out as described previously (6) except as
noted in the text. Concurrent synthesis and hydrolysis
of polydeoxythymidylate was measured under the conditions for
assay of 5' -+ 3' exonuclease, but [aH]d(T)~~ was substituted for
[aH]d(T)a~~, and [C~-~P]~TTP was present at 0.5 mM.
Endonuclease AssaysEndonuclease assays were carried out
in 67 mM potassium phosphate (pH 7.4) and 6.7 m~ MgCll using
one of the following substrates; ['4Cld(A) 4000 9 [aHld(T) 4000; ["CI-
d(A)tooo. [3Hld(T)4~~~ (7 % fi) ; and P4C1d(A)4ooo. PHI~(T)zoo.
The dA and dT concentrations were 30 p~ and 26 p~,
respectively. After 30 min at 37", the assay mixture was made
10 mM in EDTA to stop the reaction, and 100 pl were layered on
a 4-ml alkaline sucrose gradient (5 to 20 % sucrose in 0.1 M NaOH,
0.9 M NaC1). The gradients were centrifuged at 50,000 rpm for
6 hours at 4" in a Spinco L-2-65B centrifuge with an SW56 rotor.
Fractions of 10 drops (0.1 ml) were collected and counted in
Triton-toluene scintillation fluid (19).  Endonucleolytic cleavage
was estimated by comparing the sedimentation profile of the
polynucleotides in the assay to those of untreated polynucleo-
tides sedimented in parallel gradients. Since the labeled
polynucleotides are somewhat heterogeneous, the assay is not
very sensitive, but one endonucleolytic break in 50% of the
polynucleotide molecules could have been detected.
Synthesis of [14Cld(C)lIzcr[aH]d( T) IsrThe block copolymer
d(C) 1t0-d(T)160 was prepared by synthesizing d(C)lto first and
then extending this chain with deoxythymidylate residues using
terminal transferase.  [14C]d(C)lz~ was synthesized in 1 ml of 100
mM cacodylate, 100 mM potassium phosphate (pH 6.8) contain-
ing 0.25 mM CoCb, 0.67 m~ [WIdCTP, 16 pad d(C),, and 54 pg
of terminal transferase.  The polymerization was complete
(>W% utilization of the [WIdCTP) after 8 hours at 37", and
the reaction wm stopped by addition of 25 pl of 45% KOH.
After 10 min at 4" the mixture was neutralized with 20 pl of 85%
&PO4 and dialyzed for 36 hours against 1 M NaCl, 10 mM po-
tassium phosphate (pH 7.4), and l mM EDTA.  This deoxy-
cytidylate polymer (calculated to be d(C)uo) was extended with
deoxythymidylate residues in a reaction mixture identical with
that described above but containing 0.5 mM ['HIdTTP, 0.38 mM
d(C)120, and 36 pg of terminal transferase.  Polymerization was
complete in 6 hours with >95% utilization of the dTTP; the
reaction was stopped and the polymer purged as described
above. The theoretical composition of the block copolymer is
d(C)Imd(T)1so, and this value agrees with the ratio of ["C] to
Further purification of the d(C)lro-d(T)lso was effected by
annealing the polymer to an excess of d(A)rooo and sedimenting
the duplex in a neutral sucrose gradient (5 to 20% sucrose in 0.1
M NaC1,lO mM potassium phosphate bder (pH 7.4), and 1 mar
EDTA) as described above. Of the dC residues, 7% did not
sediment with the d(A)4000, but remained near the top of the
gradient in a region which had a mole ratio of dC to dT of 15;
these fractions were discarded.  The peak fractions further
down the gradient (dC to dT mole ratio = 0.75) were pooled and
concentrated. The d(C) 1zo-d(T)Iso was separated from the
d(A)tooo by sedimentation in alkaline sucrose as described above.
This is the preparation of d(C)Izo-d(T)160 used in the nuclease
assay.
The copolymer was also tested for the presence of thymidylate
strands which contained only a few cytidylate residues at the 5'
end by annealing to d(1) 1000 (20) and sedimenting the mixture in
a neutral sucrose gradient.  Of the dT residues, 6% remained
at the top of the tube in a region with a molar ratio of dC to dT
of 0.1. However the copolymer was not purified using this
method due to our inability to separate completely all of the dI
strands from the copolymer; introduction of this small amount
of oligo(d1) would have complicated assays using the block
copolymer.
Proteolytic Cleavage of DNA Polymerase and Separation of
Fragments-DNA polymerase was split into two fragments by
proteolytic cleavage as described by Klenow and Overgaard-
Hansen (3). Of intact DNA polymerase, 6.0 mg were incubated
in 20 ml containing 67 my potassium phosphate (pH 6.5), 25
mM EDTA, 30 mM (NH& sod, 10% glycerol, 3 mg of BSA, 6.25
mg of activated calf thymus DNA, and 20 I.cg of subtilisin
(Carlsberg). After 40 min at 37", the mixture was chilled in ice
and then 0.2 ml of 33 m~ PMSF was added to inactivate the
subtilisin (21). After 1 hour at 4", the mixture was adjusted to
0.2 M in potassium phosphate (pH 6.8), 1 mM in @-mer-
captoethanol, and DNA was removed by passage of the enzyme
through a 5-ml column of DEAE-cellulose equilibrated in the
same buffer. The fraction passing through the column was
dialyzed for 24 hours against two changes of 20 mM potassium
phosphate (pH 6.8), 1 m~ j%mercaptoethanol, and then adsorbed
to a 35-ml phosphocellulose column equilibrated in the dialysis
buffer. The two fragments were separated by a hear gradient
of 0.04 M + 0.16 M potassium phosphate (pH &S), 1 mM @-mer-
captoethanol (100 ml of each buffer). Appropriate column
in the final copolymer.
234 DNA Polymerase: Two Distinct Enzymes in One Polypeptide. II Vol. 247, Xo. 1
148
16
27
260"
fractions were pooled and concentrated by vacuum dialysis.
Total recoveries of both fragments were 70% (see Fig. 1).
Chronmtography-Degradation products of [aH]d(T)p~~ and
[*H]~(T)zoo (7% T^T) were separated by descending chromatog-
raphy on DE&-paper by sequential elution with 0.25 M ammo-
nium bicarbonate and 0.3 M ammonium formate (9).  Degra-
dation products of [fit y-a2P]pppTp(T)~~~ (;&Tp(T)zoo) were
chromatographed for 40 hours on Schleicher and Schuell orange
ribbon paper with markers of dTTP, pppTpT, and pppTpTpT.
The solvent system was isobutyric acid-1 M NHrOH-0.1 M EDTA
(100:60:1.6) (11).  Strips of 5 mm were cut out and counted.
Miscellaneous-Thymine dimers (TT) were formed in oligo-
and polynucleotides of deoxythymidylate by irradiation with a
low pressure mercury lamp.  The absorbance at 267 nm was
followed with time, and the percentage of thymine residues con-
h
<3
<0.2
37
37b
Fracti  on^ num ber
FIG. 1. Separation of the active fragments of DNA polymerase
produced by proteolytic cleavage of intact DNA polymerase.
Details of the cleavage and phosphocellulose chromatography
are under "Methods."  The gradient was started at fraction
number 4, and 3-ml fractions were collected.  The 5` -.) 3` exonu-
clease unit is 10 nmoles of nucleotide excised per hour.
TABLE I
  Catalytic activities of small fragment compared with
           intact enzyme
DNA synthesis was measured with calf thymus DNA, and
5' 4 3' exonuclease with concomitant DNA synthesis was as-
sayed on d(A)~oo~~[*H]d(T)z~~i with 0.5 mM dTTP.  Enzyme con-
centrations were determined from the absorbance at 278 nm.
DNA synthesis.. ...................
3' -+ 5' Nuclease. ..................
5' 4 3' Nuclease. ..................
5' -+ 3' Nuclease in the presence of
deoxynucleoside triphosphates. ...
0 5` 4 3' Nuclease concomitant with DNA synthesis.
a No DNA synthesis.
verted to dimers was calculated from the data of Deering and
Setlow (22).
SDS-acrylamide gels were run as previously described (1) and
protein molecular weights were determined on these gels by the
method of Shapiro et al. (23) with the modifications of Weber
and Osborn (24).  DNA polymerase, bovine serum albumin,
the dimer of bovine serum albumin, ovalbumin, and chy-
motrypsinogen were used as molecular weight markers.
RESULTS
Isolation of Small Fragment
Two laboratories have demonstrated that proteolysis of E. coli
DNA polymerase yields an active fragment (the large fragment)
of molecular weight 76,000 which retains the polymerase and
3' -+ 5' exonuclease activities of the intact enzyme (1, 2).
Klenow and Overgaard-Hansen have further demonstrated that
if the proteolysis is carried out in the presence of DNA, the po-
lymerase is cleaved and one obtains not only the large fragment,
but also a small fragment (mol wt 36,000) which contains only the
5' + 3' exonuclease activity (3).  We have confirmed this result
and have separated the two fragments by phosphocellulose chro-
matography (Fig. 1).  Each of the purified fragments gave a
single band on SDS-acrylamide gel electrophoresis and was
stable for at least 6 months when stored in liquid nitrogen.  How-
ever, the small fragment was quite labile at 37", and lost over 50%
of its activity in 25 min under assay conditions; in contrast, both
the large fragment and the intact enzyme are stable under these
conditions?
Catalytic Activities of Small Fragment
The small fragment had full 5' 4 3' exonuclease activity on a
double-stranded DNA substrate, with even a slightly higher
turnover number than that of the intact enzyme (Table 1).
However, the small fragment had no detectable nuclease activity
on single-stranded DNA and no detectable polymerase activity
(Table I).
The 5` + 3' exonuclease of the intact enzyme was dramatically
stimulated by concomitant DNA synthesis (Table I) (8).  This
effect was, as expected, not seen with the small fragment (Table
1).
Specijieity of Small Fragment
Degradation Products of d(A)4000'd(T)200 and d(A)4000-d(T)~,o
(7% TT)-The 5' + 3' nuclease of the intact enzyme generated
a characteristic mixture of mono- and oligonucleotides upon
degradation of d(A)dooo .d(T)zoo.  The distribution of products
was unaffected by the extent of hydrolysis of the substrate (Table
11) (8).  The small fragment also produced the same distribution
of mono- and oligonucleotides and exhibited the same specificity
as did the intact enzyme (Table 11).  The products were also
similar when the 5` + 3' nuclease action was on a d(A)4000.
d(T)200 in which 7 % of the thymine residues were linked thymine
dimers.  Both the intact enzyme and the small fragment de-
graded this substrate to about the same extent, and excised the
same amount of thymine dimers as oligonucleotides of 4 to 8
residues in length (Table 11) (9).  Larger oligonucleotides do not
migrate from the origin in the chromatographic system used
and were therefore not detected.
A
2 P. Setlow and A. Kornberg, unpublished results.
Issue of January 10, 1972
P. Setlow and A. Kornberg
235
130
TABLE I1
-.-
Degradations were carried out at 37" in 0.4 ml of 67 mM potas-
sium phosphate (pH 7.4), 6.7 m~ MgCL, and 67 fig per ml of BSA
containing 32 nmoles of d(A)mo and 28 nmoles of either [aH]-
d(T)eoo or [aH]d(T)~o~ in which 7% of the residues were thymine
dimers.  Intact polymerase (40 pmoles) or the small fragment
(48 pmoles) was added to start the reaction; loO-pl samples were
removed and hydrolysis was halted by addition of 3 pl of 0.5 M
EDTA.  The digest was chromatographed on DEAE-paper with
appropriate markers.  The percentage of degradation of the
d(T)loo was determined by absorption of a small sample (10 pl) of
the digest to DEAE-paper, and elution of mononucleotides and
smdl 01igonucleot.ides with 0.3 M ammonium formate (pH 8.5).
I
__--
d (T) I
d (TI :
d(T)a
tl (T)<:8
~____
%        %       %      %
79     79-81      72     71
17-18     16-17      16     13
3-4        3      <1     <1
<1       (1       13'     16"
I
Srnoll Fragincnt
~Is~/lOa J wactwn
2300" <0.3b
480" <O.lEid
27OC I <0.15d
pmolss/lW rJ reaction
2100" <0.25b
    <0.15d
!:: I <0.15d
0
Q
M
0
N
   Intact
DNA Polymeraso
PPP'P-i,
Distance migrated- cm
FIG. 2. A dinucleoside tetraphosphate as the degradation
product of d(A)rooo.IfhpTp(T)*~~.  The reaction was carried out
in 70 pl of 67 mM potassium phosphate (PH 7.4), 6.7 IRM MgCIz,
67 pg per ml of BSA containing 32 nmoles of d(A)4000,28 nmoles of
lffipTp(T)*oo, and either 22 pmoles of intact polymerase of 18
pmoles of the small fragment.  After a M-min incubation at 37"
the reaction was halted by addition of 2 pl of 0.5 M EDTA, and
then 504 were chromatographed in isobutyric acid-1 M NH40H-
0.1 M EDTA (100:60:1.6).
     TABLE 111
Lack of endonuclease activity
The assay conditions were described under "Methods," and
both endonuclease and exonucleolytic hydrolysis were measured.
The intact enzyme and the small fragment were both assayed at
240 pmoles per ml.  This gives a ratio of enzyme to 3' or 5' ends
of 1.8 to 1 with the substrate d(A)4000.d(T)too, and a ratio of 36 to
1 with the other substrates.
Substrate
     Nuclease breaks
Intact enzyme I ~rna11 frngment
Exo-    Endo-    Ex-    Endc-
nuclease I  nuclease 1 nuclease I  nuclease
Greater than 90% of the hydrolysis was of the dT strand.
Approximately 50% of the hydrolysis was of the dT strand.
b Measured only on dA strand.
d Measured on both dA and dT strands.
236 DNA Polymerase: Two Distinct Enzymes in One Polypeptide. II Vol. 247, No. 1
a
5chemeI: Possible Sites for Endonuclease Cleavage
C
d
          TABLE IV
Lack of hydrolysis of d(A)4Ooo'd(C)l~O-d(T)leO
The reactions were carried out at 37" in 200 pl of 67 mM sodium
phosphate (pH 8.0) ,67 m~ MgClg containing 30 nmoles of d(A)tooo,
13.4 pg of BSA, and either 2.0 nmoles of [sH]d(T)eoo or 2.8 nmoles
of [14C]d(C)~~~-[8H]d(T)~ao.  Prior to addition of the BSA, the
mixture was heated to 80" for 5 min and then quickly cooled in
ice to destroy the secondary structure of the deoxycytidylate
strands; 14 pmoles of small fragment were added to each assay
and 1.4 nmoles of [W]d(C)m were added where noted.
Hydrolysis
Substrate
1 dC I dT
might be more frequent with the small fragment which may have
lost the ability to orient itself through binding to a 3'-OH termi-
nus. Although these endonucleolytic reactions are only possi-
bilities, the importance of endonuclease action in proposed
models of DNA replication and repair (5, 27, 28) make their
assay in both the intact enzyme and the small fragment impor-
tant.
The endonuclease activity of both the small fragment and the
intact enzyme was therefore tested on the several different sub-
strates shown in Scheme I (a, b, c).  The ratio of enzyme to
available nicks or ends was varied from 2 to 36, but no endonu-
clease was detected. The ratio of measured exonucleolytic
cleavage to the upper limit of endonucleolytic cleavage was sl-
ways greater than 108 in experiments in which the extent of
exonucleolytic degradation of the substrates varied from 2 to
80% (Table 111).
Lack of Degradation of Long Displaced Oligonucleotide-The
data in the previous section suggest that whereas the 5' + 3'
exonuclease activities of both the intact enzyme and the small
fragment can excise small oligonucleotides, they are unable to
excise large oligonucleotides.  This was tested directly using the
substrate d(A)4aoo.d(C)lzO-d(T)l60 (Scheme Id). When the
small fragment was assayed on this substrate under conditions
where poly (dC) has no secondary structure (20) there was no
detectable degradation of the dC strand; also <2% of the dT
was solubilized (Table IV).  The rate of solubilization of thy-
midylate residues from this substrate was also more than 40
times slower than the same reaction with the substrate d(A)tOoO.
d(T)d (Table IV).  Similar results were obtained using the
intact enzyme, but the experiment was complicated by the action
of the 3' + 5' exonuclease.
The data in this and the previous section suggest two con-
clusions concerning 5' + 3' exonuclease action: (a) the 5' + 3'
exonuclease of both the intact enzyme and the small fragment
cannot act as an endonuclease since there is an upper limit to the
size of the oligonucleotide that can be excised, and (b) the speci-
ficity of the 5' -t 3' exonuclease is similar in both the intact en-
zyme and the small fragment.
Cooperative Interaction of Mixture of Small
     and Large Fragments
There are three reactions in which the catalytic properties of
the intact enzyme are strongly influenced by concomitant action
of both the 5' + 3' exonuclease and polymerase activities: (a)
stimulation of 5' -+ 3' exonuclease by concurrent DNA synthesis
(8), (b) the excision of oligonucleotides greater than 3 residues in
length (8), and (c) kinetics of primed net synthesis of the copoly-
mer poly[d(A-T)] (1, 6).  The following experiments show that
these properties of the iiitact enzyme can be reconstituted in
mixtures of the small and large fragments of DNA polymerase.
Stimulation of 6' + 3' Exonuclease by DNA Synthesis-The
stimulation in 5' --+ 3' exonuclease activity by DNA synthesis
has been attributed to concomitant action of the polymerase and
5' + 3' exonuclease activities of a single enzyme molecule (8).
If the two separate fragments bind next to one another at the
same nick in a double-stranded DNA, the adjacent fragments
might then act as a single enzyme.  Therefore the 3' and 5'
termini of the substrate d(A)tooo.d(T)200 were saturated with the
large and small fragments, respectively, and 5' + 3' exonuclease
activity was measured (Table V).  Under these conditions, the
small fragment was stimulated by simultaneous DNA synthesis
by the large fragment.  The turnover number of the stimulated
exonuclease was only slightly less than the same reaction cata-
lyzed by the intact enzyme (Table V): Furthermore, under
these conditions of saturating levels of both fragments, nick
translation in which hydrolysis of DNA matches synthesis was
observed (Fig. 3) as with the intact enzyme previously (8).
Path of Oligonucleotide Excision during DNA Synthesis
Additional evidence for the coordinated action of the catalytic
activities of the two fragments was observed when the products
a This number may be even higher, since the slow rate of solu-
bilization of thymidylate from the block copolymer may be due to
degradation of the small percentage of thymidylate strands which
have only a few dC residues at the 5' end (see "Methods").
Issue of January 10, 1972
Small fragment
None. ...................................
dTTP. ...................................
None. ...................................
dTTP ....................................
None. ...................................
dTTP ....................................
Small fragment + large fragment
Intact enzyme
P. Setlow and A. Kmberg
males nuclwiidc ~iSCdl
mole snrymdman
     37
     37
     33
     188
     27
    260
237
d(T)i
d(Va
d(T)~-s
d (TI z
TABLB V
6' 4 3' Exonuclease activity during concomitant DNA synthesis
The assays were carried out in 0.2 ml as described under "Meth-
ods."  To each assay, 3.0 nmoles of d(A)4000 and 2.7 nmoles of
[aH]d(T)~~~ were added; 14 pmoles of the small fragment were
present and 18 pmoles of the large fragment were added.  dTTP
was added to 0.5 mM.
    % %
55 71 47 75
22 29 23 25
10 <1 11 <1
13 <1 19 <1
Enzymes and addition
I 5' 4 3' Exonuclease
I
C
I-
-5
I
Small Crcrqrnant +
Largo Crsgmanf
v)
v)
c)
.-
e hydrolysis
0 Jynthesis
2 4
Time in minutes
FIG. 3. Nick translation, the simultaneous synthesis and hy-
drolysis of DNA, catalyzed by a mixture of small and large frag-
ments.  The incubations were carried out as described in the
legend to Table V, but with [or-a*P]dTTP at 0.5 m.  Twenty-five-
microliter samples were withdrawn from the assays with time,
and adsorbed to squares of DEAE-paper for measurement of
both synthesis and hydrolysis of polydeoxythymidylate.
generated by 5' -+ 3' exonuclease action during DNA synthesis
were analyzed.  Under these conditions the intact enzyme ini-
tially excised a high percentage (13%) of oligonucleotides of 4 to
8 residues in length (Table VI) (8), while in the absence of DNA
synthesis less than 1 % of the products were large oligonucleotides
(Table 11).  The small fragment also produced no large oligo-
nucleotides (Table 11), and the product distribution was not
&Teated by addition of either the large fragment or deoxynu-
cleoside triphosphates?  However a mixture of both fragments
plus deoxynucleoside triphosphates produced a high percentage
          TABLE VI
Products of hydrolysis of d (A)4000*d(T)200 during
     concomitant DNA synthesis
The degradation and the chromatography of the digests were
carried out as described in the legend to Table 11, but with only
16 nmoles of d(A),ooo and 14 nmoles of [SH]d(T)roo.  The intact
enzyme was present at 150 pmoles per ml, the small fragment at
220 pmoles per ml, and the large fragment at 270 pmoles per ml;
0.5 mM dTTP was added.
Products
intact enyme + dm
Lar e fragment + small
1 &gment+dTTP
I hydrolysis 35% I hydro 1WF. YSIS 1 hydrolysis 25% 1 hydrolysis 98%
/OO % utilization OC dTTP-
i
I I I
550 1000 1500
Time in minutm
FIQ. 4. Primed poly[d(A-T)] synthesis by the large fragment
or a mixture of the two fragments.  The reaction WBB carried out
at 37" in 2.5 ml of 67 mM potassium phosphate (pH 7.4), 67 m~
Malr, 75  dATP and 75 p~ [*H]dTTP.  BSA was present at 100
pg per ml and poly[d(A-T)~oo~] at 0.7 p~. The reaction
was started by addition of the large fragment (18 pmoles) or a
mixture of the large fragment (18 pmoles) and the small fragment
(20 pmoles).  Poly[d(A-T)] synthesis waa measured by adsorbing
a 4O-pl sample of the reaction mixture to squares of DEAE-paper
and eluting unreacted triphosphates and oligonucleotides with
0.3 M ammonium formate (9).  The symbols in the inset are the
same as those in the main figure.
(19%) of large oligonucleotides.  After 3 hours at 37O, when
hydrolysis wm complete, both the intact enzyme and the mixture
of fragments eventually degraded the large oligonucleotides to a
mixture of mono- and dinucleotides (Table VI) (8).
Kinetic8 of Primed PoZy[d(A-T)] Synthesis--Another reaction
which involves the participation of both polymerase and 5' --t 3'
238 DNA Polymerase: Two Distinct Enzymes in One Polypeptide. 11 Vol. 247, No. 1
Large fragments
exonuclease is the primed synthesis of poly[d(A-T)].  The ki-
netics of the reaction catalyzed by the intact enzyme is
autocatalytic early in the synthesis.  By contrast the reaction
catalyzed by the large fragment is relatively linear and also less
rapid (Fig. 4) (1). Since poly[d(A-T)] synthesis is carried out
with a 20-fold excess of polymerase molecules over 3'-OH primer
ends, a possible explanation for the autocatalytic kinetics with
the intact enzyme is the generation of poly[d(A-T)] oligonucle-
otides by action of the 5' + 3' exonuclease. These oligonucle-
otides might act as new primer strands, which the large excess of
polymerase molecules could utilize for synthesis of poly[d(A-T)].
Such a mechanism for increasing the rate of poly[d(A-T)] synthe-
sis would not be available to the large fragment for lack of 5' + 3'
exonuclease activity.
In agreement with these observations, the more rapid
autocatalytic mode of poly[d(A-T)] synthesis was restored to the
large fragment by addition of an equimolar amount of the small
fragment (Fig. 4).  Presumably concomitant action of the two
fragments caused excision of oligonucleotides of poly[d(A-T)]
(see Table VI), which acted as new primer strands.  In poly-
[d(A-T)] synthesis catalyzed by the intact enzyme, the 5' -+ 3'
Theoretical value
Small fragments
500
- 400
E
t
--.
0
S
3
PO0
>
+
0
(3
u
ln
1
.-
.-
? 200
E
n
-
0
Q
IO0
0
2.50
0.50
0.16
I
,
Sedi rncntation
R
0.80 355
0.80 1966
0.30 123 116
0.12 70 61
Fraction number
exonuclease eventually degrades all the product.  This was not
the case with the mixture of the two fragments presumably
because the lability of the small fragment at 37" led to
denaturation of the 5' -+ 3' exonuclease activity early in the
reaction?
Lack of Direct Binding between Small and
     Large Fragments
The preceding experiments demonstrated that a mixture of the
small and large fragments of DNA polymerase in the presence of
DNA restores the properties of the intact enzyme.  This result
might be caused by reassociation of the two fragments on the
DNA or independently of it.  Binding of one fragment to the
DNA may or may not favor binding of the other fragment adja-
cent to it.
Inasmuch as the large and small fragments are readily sepa-
rated by Sephadex (3) or phosphocellulose chromatography
(Fig. l), afKnity between them cannot be strong.  This was
demonstrated more directly by preparing a mixture of the frag-
ments and then sediienting them on a sucrose gradient (Fig. 5).
The small and large fragments sedimented independently of one
another; their positions in the gradient were identical when they
were sedimented alone or together.
Similarly, the separate fragments exhibited no physical affinity
for one another on DNA (Table VII).  The turnover number of
the small fragment was measured in the presence of deox-
yribonucleoside triphosphates and with several ratios of large
and small fragments to 3' and 5' ends on the DNA.  At a high
level of saturation of the 3' and 5' ends, the two fragments bound
adjacent to one another at the same nick, and the result was
similar to that seen in Table V.  The turnover number of the
small fragment was increased by the concomitant DNA synthesis
of an adjacent large fragment.  However, the turnover number
           TABLE VI1
Lack of afinity between small and large fragments
            on DNA
Assays were carried out aa described in Table V with different
amounts of both fragments as noted and with 0.5 mM dTTP.
Turnover number gf 5' - 3'
exonuclease activity"
Ratio of fragments to 3' or 5' ends
FIG. 5. Sedimentation of a mixture of the large and small
fragments in a sucrose gradient.  The two fragments were mixed
in 67 m~ potassium phosphate (pH 7.4), 0.2 mM p-mercaptoethanol,
and incubated at 37' for 10 min and then at 4' for 50 min.  One
hundred microliters of this solution containing 300 pmoles of the
large fragment and 100 pmoles of the small fragment were layered
on a 4-ml sucrose gradient (5 to 20% sucrose, 67 mM potaesium
phosphate (pH 7.4), 0.2 m~ 8-mercaptoethanol) and centrifuged
at 4" in a SDinco model L2 66B centrifuge with a SW 56 rotor at
56,OOO rpm.- After 12 hours, 20-drop fractions were collected and
assayed for polymerase and 5' + 3' exonuclease activities.  In-
tact enzyme (300 pmoles), the large fragment, and the small
fragment were also sedimented as markers in three parallel gradi-
ents. during DNA synthesis.
Nanomoles of nucleotide excised per mole of small fragment
per min.
* These values are calculated assuming that the small and large
fragments bind both very tightly to 5' and 3' ends, respectively,
and that the binding is uninfluenced by the presence of another
fragment. A value of 196 waa found for asmall fragment adjacent
to a large fragment and 35 for a small fragment alone.  Thus with
a 0.50 ratio of large fragment, one-half of the small fragments
would have a neighboring large fragment (196/2 = 98) and one-
half would not (35/2 = 18).
0 Specific activity of small fragment alone.
d Specific activity of small fragment adjacent to large fragment
of the small fragment decreased as the ratio of fragments to 3'
and 5' ends decreased; the experimental values were almost
identical with values calculated assuming random binding of the
fragments to DNA.  If the fragments had had a strong affinity
for one another on DNA, a decrease in the ratio of fragments to
3' or 5' ends would not have resulted in a corresponding decrease
in the turnover number of the small fragment.
DIBCUSSION
Efects oj DNA Synthesis an Excision FunctionStudies of the
action of the separate fragments of DNA polymerase suggest a
mechanism whereby concomitant DNA synthesis could both
stimulate the rate and alter the specificity of the 5' -+ 3' exonu-
clease.  The large fragment is fuUy active in catalyzing DNA
synthesis on a nicked double-stranded DNA and probably dis-
places the strand ahead of the 3'-OH growing point (6).  It is
therefore probable that DNA synthesis by the intact enzyme
may also cause some fraying at the 5' end of the strand ahead of
a growing point.  This frayed strand might be more susceptible
to degradative attack by the same enzyme molecule (8) resulting
in an increased rate of 5' 4 3' exonuclease.  The fraying of the
5' end will also explain the excision of oligonucleotides (Table VI)
since both the intact enzyme and the small fragment are known
to excise large oligonucleotides from a DNA duplex containing
frayed 5' ends produced by thymine dimer formation (Table 11)
or the introduction of mismatched bases (9).  The reason for the
apparent upper limit on the size of the oligonucleotides produced
by 5' -+ 3' exonuclease action is, however, not clear.
Concomitant Action of Small and Large Fraction-The stimu-
lation in the exonucleolytic rate of the small fragment by con-
current DNA synthesis catalyzed by the large fragment can best
be explained by concomitant action of two different fragments
which have bound adjacent to one another at a single nick in a
DNA molecule.  The alternative explanation that DNA syn-
thesis by the large fragment causes displacement of the 5' end
which is then attacked by an unbound small frapent Seems less
likely since the small fragment shows an absolute specificity for
a double-stranded substrate, although the 5' end at which the
small fragment acts may have some single stranded character,
i.e. may be frayed due to a thymine dimer or some other cause.
Previous work of Kelly et at. indicated that the stimulation in
5' + 3' exonuclease by DNA synthesis in the intact polymerase
also involved concomitant action of the polymerase and 5' -+ 3'
exonuclease activities of the same molecule (8).
Lack of Afinity between Small and Large Fragment-The exper-
iment demonstrating the lack of aEnity between the two frag-
ments in the absence of DNA is quite conclusive (Fig. 5), but
demonstration of their lack of &nity in the presence of DNA
(Table VII) is based on two assumptions: (a) that the two frag-
ments bind tightly to DNA, and (b) that the binding of the
fragments to DNA is reversible and allows formation of
thermodynamically favored states. Sedimentation studies
have demonstrated that intact DNA polymerase binds tightly to
both DNA and poly[d(A-T)] oligomers (29), and we have similar
data for the binding of the large fragment to d(A)4000-d(T)200.2
The small fragment also binds tightly to DNA, since the rate of
5' -+ 3' exonuclease on d(A)cooo.d(T)200 was the same when the
ratio of small fragment to 5' ends was reduced from 3 to 1 to 1 to
1.  There are no data on the dissociation rates of the individual
fragments from DNA, but the binding of the intact enzyme to
Issue of January 10, 1972
P. Setlow and A. KomEerg
239
16. SCHACHMAN, H. K., ADLER, J., RADDING, C. M., LEHMAN,
DNA is reversible (29).
There are no data on the velocity of
I. R., AND KORNBERG, A. (1960) J. Biol. Chem., 2!J6,3242.
this dissociation, but it takes place at 4O, while our experiments
were performed at 37" where the dissociation would be expected
to be more rapid.
Structure of DNA Polymerase-The cleavage of DNA
polymerase into two active fragments which show no affinity for
one another, and the similarity of their catalytic reactions to the
analogous reaction catalyzed by the intact enzyme indicates
that within the single polypeptide chain of DNA polymerase are
two distinct enzymes, one enzyme containing the polymerase and
3' --+ 5' exonuclease function and the other enzyme containing
the 5' -+ 3' excision function. The two enzymes are held to:
gether by a polypeptide link (susceptible to proteases), which
ensures that both the polymerase and excision function act si-
multaneously at the same nick in a DNA molecule.
Possible Importance of Two Enzymes-Single Polypeptide
Structure-The coupling of the polymerase and excision func-
tions in a single enzyme may be of significant advantage in recom-
binational events and in the in Vivo repair of lesions in DNA
such as thymine dimers.  As a thymine dimer is excised by the
excision function, concurrently the polymerase function fills in
the gap caused by the excision.  Therefore excision repair by
DNA polymerase at no time leaves a single strand gap which
might be attacked at the 3' end by exonucleases such as exonu-
clease 111, but rather leaves only a nick with a 3'-OH and a 5'-P
from which the polymerase can be displaced by DNA ligase.
Possibly the extensive DNA degradation observed after
ultraviolet irradiation of E. coli mutants which lack DNA
polymerase (pol A mutants (30)) is the result of the production
of single strand gaps during the excision of thymine dimers by
secondary repair systems (31, 32). The lifetime of these gaps
may be long enough to enable enzymes such as exonuclease I11
to attack these single strand gaps and thereby cause extensive
degradation of the DNA.
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