THE JOURNAL or BIOLOQICAL CHEUIST~Y
Vol. 244, No. 11. Issue of June 10,pp. 3WW-3018, 1969
         PrinluI in U.S.A.

Enzymatic Synthesis of Deoxyribonucleic Acid

XXVII. CHEMICAL MODIFICATIONS OF DEOXYRIBONUCLEIC ACID POLYMERASE*

(Received for publication, November 6, 1968)

THOMAS M. JOVIN,$ PAUL T. ENGLUND,~ AND ARTHUR KORNBERG
From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 942 06

SUMMARY

The purified Escherichia coli DNA polymerase contains a
single sulfhydryl group which reacts with Hg(I1) to produce
either a monomer contahing a single mercury atom or a dimer
of two protein molecules joined by a mercury atom.  Both
forms retain full polymerase and exonuclease activities, im-
plying that the sulfhydryl group is relatively exposed and not
essential for enzymatic activity.

The acylating agent, N-carboxymethylisatoic anhydride,
reacts with an observed maximum of 11 sites on the enzyme
to form a highly fluorescent derivative with altered functional
properties.  Only 0.2% of the original polymerase activity
but 920% of the exonuclease activity are observed at pH 7.4
with DNA as primer or substrate.  Measurements of release
of inorganic pyrophosphate and binding of triphosphates indi-
cate that the predominant effect of this acylating agent is to
cause a marked reduction in the affinity for deoxyribonucleo-
side triphosphate substrates, but concomitant changes in the
interaction of the enzyme with polynucleotides are probably
also involved.  The data are discussed in terms of a model in
which the several activities of the enzyme are catalyzed at
the same active center, which is differentiated into physically
distinct sites.

In the preceding paper of this series (1) some of the physical
and chemical characteristics of the purified Escherichia coli
DNA polymerase were presented.  This communicaticn will
describe the effects of two chemical modifications on the physical
and chemical properties of the enzyme.  One of these, the addi-
tion of a mercury atom to the single sulfhydryl group of the
enzyme, does not affect any of its activities, and with the radio-

* This study was supported in part by grants from the National
Institutes of Health (United States Public Health Service), the
National Science Foundation, and the National Aeronautics and
Space Administration.
3 National Science Foundation Postdoctoral Fellow.  Presently
Established Investigator, American Heart Association; present
address, Max Planck Institut fur physikalisehe Chemie, Got-
tingen, Germany.

$ National Institutes of Health Postdoctoral Fellow.  Present
address, Department of Physiological Chemistry, The Johns
Hopkins University School of Medicine, Baltimore, Maryland
21205.

isotope, 20*Hg, provides an extremely sensitive tracer for physical
studies.  The other modification is the production of a fluores-
cent derivative with the acylating agent, N-carboxymethylisatic
anhydride.  This derivative has useful properties for physical
studies, and is the first example of a differential modification of
the enzymatic activities associated with the DNA polymerase
molecule.  The virtual abolition of polymerase function with
retention or even enhancement of the exonuclease activity,
coupled with the indications that a single enzyme center encom-
passes both functions, provides additional information about the
active sites of the enzyme.

EXPERlMENTAL PROCEDURE

Materials

20sHg(N03)2 (in 2 N HNOa), obtained from New England
Nuclear, was diluted to about 2 mM and neutralized with Tris.
The Hg(I1) concentration was determined by a titration of re-
duced glutathione based on the 5,5'-dithiobis(2-nitrobenzoic
acid) sulfhydryl method of Ellman (2); 0.5 mole of Hg(I1)
neutralized the reactivity of 1.0 mole of glutathione for 54'-
dithiobis(2-nitrokenzoic acid).  The specific radioactivity, deter-
mined with a scintillation counter set for 14C counting, was 1.6
x 108 cpm pmole-1.  The synthesis and properties of NCMIA:
a gift of Dr. Lubert Stryer, will be described elsewhere.*  5,5'-
Dithiobis(2-nitrobenzoic acid) was obtained commercially.
DEAE paper was Whatman DE-81.  Darco G-60 was a product
of Atlas Chemical Industries.  DNA polymerase was the homo-
geneous Fraction 7; its concentration was based on a molecular
weight of 109,000 (1).  The sources of other materials and rea-
gents are given in the previous paper (1).

Methods

Sucrose Gradient CentrifugationSucrose gradient centrifuga-
tion was performed according to the method of Martin and Ames
(3).  Gradients (3.6 ml) of 5 to 20% sucrose in 0.2 M potassium
phosphate buffer, pH 7.4, were centrifuged at 60,000 rpm in an
International model B-60 centrifuge (SB 405 rotor) for 11 hours
at 3'. Fractions were analyzed for enzymatic activity, radio-

1 The abbreviations used are: NCMIA, N-carboxymethylisatoic
anhydride (N-carboxymethyl-N-carboxyanthranilic acid, cyclic
anhydride); poly d(A-T), the alternating copolymer of deoxy-
riboadenylate and deoxyribothymidylate previously designated
dAT.

* R. P. Haugland and L. Stryer, manuscript in preparation.

3009

3010 Enzymatic flyrunasis of DNA. XXVII Vol. 244, No. 11

None. . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . .
Glutathione, 0.02 M.. . . . . . . , . . . . . . . . . .
dATP + dTTP, 0.05 mM each.. . . . . . . .
Poly d(A-T), 0.1 mMa.. . . . . . . . . . . . . . . .

Fraction

FIG. 1. Gel filtration of polymerase allowed to react with
excess *O3Hg(II). The 50-pl sample contained 1.8 nmoles of
polymerase and 8.5 nmoles of '03Hg(II) in 0.1 M potassium phos-
phate, pH 7.0.  The protein had been incubated with the mercury
for 15 min at 0".  The column of Sephadex G-150 (74 cm X 0.07
cm2) was eluted with 0.2 M Tris-O.1 M HCI.  Fractions of 0.19 ml
were collected at a rate of 2.5 ml per hour at 4".  The column was
calibrated with blue dextran in a separate experiment.  The
polymerase and exonuclease assays were run without 2-mercapto-
ethanol.  Recovery of protein from the column was 62%.

% %
0 2
87 98
8 4
4 3

I I

n

Fraction

FIG. 2. Sucrose density gradient of *0SHg(II) allowed to react
with excess polymerase. The 7O-pl sample contained 2.2 nmoles
of polymerase and 1.06 nmoles of 20aHg(II) in 0.1 M potassium
phosphate, pH 7.0.  The protein had been incubated with the
mercury for 43 hours at 0".  Fractions of 0.14 ml were collected
for radioactivity determinations, enzymatic assay, and protein
determination.  The sedimentation coefficients were determined
by comparison with a hemoglobin marker in a separate tube.
The polymerase and exonuclease assays were run without mer-
captoethanol.  The recovery of protein from the gradient was
82%.

activity, and protein. Sedimentation coefficients were esti-
mated with human hemoglobin as a marker (4.6 S) at concen-
trations above 0.1 mg ml-l in order to preserve the tetrameric
state (4).
Fluorescence Spectra-Fluorescence emission and excitation
spectra of NCMIA and its derivatives were made with an
Aminco-Bowman spectrophotofluorometer equipped with an
RCA 1P21 photomultiplier. The emission, excitation, and
polarization measurements of the protein conjugates were per-
formed with the recording spectrofluorometer described by
Stryer (5). Microcuvette assemblies accommodating quartz
tubes with an internal diameter of 3 mm were used.  The emis-
sion spectra are uncorrected for variations in sensitivity of the
detection system with wavelength.  Calibrations for the system
have been given by Haugland and Stryer (6).  Corrected flu-
orescence excitation spectra and polarization measurements were
recorded directly with the use of a ratio amplifier (5).  In the
case of polarization measurements, the exciting light was isolated
with a Corning 7-31 filter.  All measurements were made at
5" f 1".

Other methods have already been described (I) or are given
in the table and figure legends.  Exonuclease assays were under
conditions described for exonuclease I1 (l), except where other
conditions are defined.

RESULTS

Reaction of DNA Polymerase with 208Hg(ZZ)

With excess 20aHg(II), the enzyme, after isolation by gel filtra-
tion, contained 1 mole of *NHg per mole of protein (Fig. I).  The
sedimentation coefficient of this derivative, as estimated by
sucrose density gradient centrifugation, was identical with that
of the untnodified enzyme (5.9 S), and agrees with the value of
5.6 S previously obtained by analytical centrifugation (1).

         TABLE I
Stability of polymerase-mercury linkage

The monomer and dimer were isolated by gel filtration and
sucrose density centrifugation, respectively (Figs. 1 and 2).  The
0.1-ml reaction mixtures, containing either 3.7 pmoles of monomer
or 6.2 pmoles of dimer, in 0.05 M potassium phosphate, pH 7.2,
were incubated for 10 min at 37".  They were then precipitated
with 0.2 ml of cold 10% trichloracetic acid and centrifuged for 10
min at 12,000 X g.  The supernatant fluid (0.25 ml) was removed
and neutralized with 0.7 ml of 0.8 M Tris.  Mercury released from
the protein was measured in a scintillation counter with the use
of 10 ml of the dioxane fluid.

1 Mercury liberated from

Treatment

1 Dimer

1 Monomer

a In a separate experiment, 0.1 IMI poly d(A-T) was incubated
with 1.08 p~ zOsHg(II).  Any 2ozHg-poly d(A-T) complex formed
must have been dissociated by the trichloracetic acid precipita-
tion, because the mercury was recovered quantitatively in the
supernatant fluid.  Had mercury been removed by poly d(A-T)
from the polymerase monomer or dimer, it would have appeared
in the supernatant fluid aft,er the acid precipitation.

Issue of June 10, 1969

T. M. Jovin, P. T. Englund, and A. Kornberg

3011

Since Hg(I1) is divalent, it might be expected to link polymerase
molecules in dimers, as it does with serum albumin (7).  This
possibility was realized in a reaction mixture with a 2-fold
excess of polymerase over Hg(I1) (Fig. 2).  Peak I was desig-
nated as dimer on the basis of a sedimentation coefficient of 7.9
S, compared to 5.9 S for the native enzyme, and the molar ratio
of mercury to protein of 0.55.  Peak ZI was a mixture of un-
modified polymerase and mercury-polymerase which was still in
the monomer state.  Had the time of reaction been increased,
all of the mercury might have been found in Peak I.  In another
experiment in which polymerase was incubated with mercury
for only 20 min before the centrifugation, less than 25% of the
*03Hg(II) wm incorporated into dimer.

The specific activities of the mercury-containing monomer
and dimer for both polymerase and exonuclease were the same
as those of the unmodified enzyme in each experiment.  It was
necessary to establish, however, that components of the mixture
used for assay of these activities, such as DNA, poly d(A-T), or
triphosphates, did not remove the mercury and regenerate the

0.

0

h

.-

i
" 3

V

- 0.

?
0

0

0

IIII!

FIG. 3. Absorption spectra of NCMIA and derivatives. a,
NCMIA inO.1 M potassium phosphate, pH 7.4; b, reaction product
of NCMIA in 0.1 M potassium phosphate, pH 7.4, after 23 hours
at 37" (no further spectral change was observed after 15 additional
hours); c, reaction product of NCMIA after ammonolysis in 13.3
M NHa for 21 hours at room temperature, in 0.1 M potassium phos-
phate, pH 7.4.  All solutions were 0.1 mM by reference to an
original NCMIA solution prepared by weight with the use of a
molecular weight of 239 (this value includes 1 water molecule of
crystallization).  The spectra were recorded on a Cary 14 spec-
trophotometer at 4-10".  The absorption maxima, in millimicrons,
of the three compounds were a, 243 and 317; b, 2-51 and 331; and e,
253 and 334.

FIQ. 4. Fluorescence emission spectra of NCMIA and deriva-
tives.  Spectra a, b, and c are of the compounds described in the
legend to Fig. 3.               Relative quantum
yields were calculated by dividing the peak emission intensity
by the absorbance at 330 mp.  The emission maxima, in milli-
microns, of the three compounds were a, 395; b, 412; and c, 438.

Excitation was at 330 mp.

native enzvme.  Englund, Kelly, and Kornberg have shown
elsewhere that mercury is not removed by DNA or poly d(A-T)
(8).  Furthermore, the results in Table I also indicate that the
mercury-polymerase linkage is stable in the presence of poly
d(A-T) and triphosphates, but that the mercury is quantitatively
removed by glutathione.  Since care was taken to omit a sulfhy-
dryl reagent from the assay solution and from the enzyme diluent,
it is likely that the mercury-polymerase linkage was intact during
the assay.  Jovin, Englund, and Bertsch have previously shown
that carboxymethylation of the single sulfhydryl group also does
not affect either enzymatic activity (1).

Electroqhoresis of mercury-Folvmerasq pya ra t,imm? fin- ~$-y:-
acrylamide gels showed that in Buffer System A (I), the mono-
meric form migrated with the same mobility as the unmodified
enzyme.  However, a mercury-polymerase preparation in which
the formation of dimers was favored showed, in 9% polyacryl-
amide gels, a new zone with a mobility of 0.75 relative to that of
the more prominent monomer zone.  The presence of a diffuse
zone of protein between the zones suggested that the equilibrium
between the two states was under constant readjustment during
the electrophoretic separation, possibly because of dissociation
and counter migration of Hg(I1).

Reactions of DNA Polymerase with NCMIA

Properties of NCMZA-The absorption and fluorescence
emission spectra of NCMIA (I) and some derivatives are shown in
Figs. 3 and 4, respectively.  Hydrolysis at 37" in 0.1 M potassium
phosphate, pH 7.4, caused a 9-fold increase in quantum yield and
red shifts of 14 mp in the absorption spectrum and 17 mp in the
emission spectrum.  The half-life in the same buffer at 0" was
about 30 hours when measured by changes in absorbance at 260
mp, in contrast with the reported half-life of 13 min for isatoic
anhydride at 25" (9).  The observation of time-dependent isos-
bestic and isoemissive points during the course of the reaction at
0" indicated the presence of more than two species and suggested
that a sequence of hydrolytic reactions was involved.

Ammonolysis of NCMIA also caused an 8-fold increase in
quantum yield and red shifts of 17 mp in the absorption and 43
mp in the emission spectra (Figs. 3 and 4).  NCMIA in 13.3 M
NHa had a half-life of 2 hours at approximately 20" and 1.15
hours at 28".  This relative stability is surprising inview of the re-

3012

Enzymatic Synthesis of DNA. XXVII

Vol. 244, No. 11

                TABLE I1
Properties of DNA polymerase derivatives prepared with NCMIA

Property

Preparation"

NCMIA (mM). ......
dATP (mM). ................................
Polymerase (mM). ..................................................

Moles dye per mole enzymeb. .......................................
NCMIA reacted (%). ..............................................

Stoichiometry of conjugation

Absorption spectra

A980: Atao. ..........................................................
A332:A2so ...........................................................

Fluorescence spectra

E4,7:E336d ..........................................................

Energy transfer (%)e.. ............................................
Emission anisotropy!. ..............................................
Quantum yield of conjugated dyer. .................................

Enzymatic activitiesh

Polymerase (poly d(A-T) primer) ..............
Polymerase (DNA primer). .................... ..........

..........

tio DNA primer to poly d(A-T) primer. ..............

.....................................................

a The incubations, in 40 pl, were for 40 hours at 0'.

                               The buffer
waa 0.05 M potassium phosphate, pH 7.5.  After reaction, the
solutions were diluted 6-fold and dialyzed extensively against the
same buffer.  The spectra were recorded without further dilu-
tions.

*Calculated from difference spectra (eg. Fig. 5) using an
mp value of 8 X 1oP r1 cm-' for the conjugated dye (Fig. 3c).
Owing to many uncertainties, the true values might differ by

about 30%.
c These values are subject to potentially large error from light
scattering.
d Ratio of emission intensities at the indicated wave lengths
(millimicrons), with excitation at 290 mp.
e Calculated from the excitation spectra (e.g. Fig. 6B) as de-
scribed in the text.

1 The emission anisotropy, A, is given by (Fz - F,)/(F. + W,),
where F, and Fu are the emission intensities with the analy-
zer set parallel and perpendicular to the polarizer, respectively.
r Quantum yield of fluorescence emission is defined as the ratio
of emitted to absorbed quanta.  An emission spectrum in which
energy transfer from tryptophan to a conjugated dye is observed
can be used to calculate quantum yield, Q, according to the
formula Q = K (Edyo/Etrp) (1 - T)/T, where Edrs and Et,, are

0

bH,-COO-

I

I1

1

0.50

0.043

6.6

55

0.98
0.26

1.77

0.166
0.6

47

0.006

0.42

Derivative

2

0.47
0.70
0.039

6.1

51

1.02
0.25

1.65

0.166
0.5

43

0.013
0.08
6
0.51

3

0.089

0.044

2.3

100

1.40
0.13=

0.47

0.155
0.4

0.10
0.27
2.7
1.18

24

4

0.077
0.72
0.040

2.2

92

1.44

0.13~

0.41

0.155

0.4

0.30
0.45
1.5
1.10

20

the intensities of the two peaks in the spectrum representing emis-
sion by the dye (due to excitation by energy transfer) and the pro-
tein emission (directly excited), respectively; Tis the efficiency of
energy transfer, measured from an excitation spectrum; and K
is an instrument constant (generally wave length-dependent).
The term Etrp T/(1 - 2') is a measure of the exciting energy
because it represents the change in the tryptophan emission
intensity due to energy transfer.  The value of K for the particu-
lar instrument used in this study was calculated by use of the
above equation with the data for anthraniloylchymotrypsin
(Fig. 1 of Haugland and Stryer (6)), a system in which Q was
measured directly. This value of K (0.29) was then used to
calculate the quantum yields for the polymerase-NCMIA deriva-
tives using the data given in this table.  Because the anthra-
niloylchymotrypsin and NCMIA-polymerase spectra had peaks
with similar half-widths and maxima (within 5 mp), peak fluores-
cence intensities rather than the integrated emission curves could
be used.

Expressed as ratios relative to unmodified polymerase.
Exonuclease I11 was not used in the polymerase assays (1).
iThe scale is normalized so that the value for unmodified
polymerase is 1.

port that isatoic anhydride and N-methylisatoic anhydride (10,ll)
react within 5 min under similar conditions.  Assuming the same
reaction pathway as observed with N-methylktoic anhydride
(10, ll), the most probable product of the ammonolysis of
NCMIA is N-carboxylmethylanthranilamide (11).

Fluorescence excitation spectra of the parent compound and
the products of its reaction with water and ammonia were com-
patible with the absorption spectra, although the peaks were
shiftedta longer wave lengths by an average of 8 mp, presumably
because of a varying emission intensity in this region of the
spectrum.

Spectral Properties of NCMIA-DNA Polymerase Conjqdes-

Issue of June 10,1969

T. M. Jowin, P. T. England, and A. Kornberg

3013

DNA polymerase was allowed to react with NCMIA at neutral
pH and 0".  The reisolated protein had spectral properties
consistent with the covalent attachment of the dye.  In one
experiment, polymerase was incubated with two levels of NCMIA
in the presence and in the absence of dATP (Table 11).  The
absorption spectra of two of these derivatives are shown in Fig.
5.  The dserence absorption spectra had peaks at 254 and 334
mp, and were similar to the spectra for the reaction product of
NCMIA and ammonia (Fig. 3).  The degree of conjugation was
a function of the input concentration of NCMIA and of the time
of incubation; the kinetics of conjugation will be described below.
The number of dye molecules per protein molecule was calculated
by using a molar extinction coefficient for the conjugated NCMIA
of 8 x los M+ cm-1 at 254 mp (Fig. 3c).  These values and other
data for the derivatives are given in Table 11.  The extent of
reaction of NCMIA at the lower concentration was 90 to 100%;
at the higher concentration it was 50 to 60%.  As a test for the
covalent nature of the attachment, the products were subjected
to electrophoresis on a 9% polyacrylamide gel with Buffer
System A.  Fluorescent zones were detected which corresponded
with the positions of the protein zones.  Derivatives 3 and 4
showed somewhat skewed distributions favoring the less mobile
protein; the center of mass had a mobility about 5% greater than
that of the control enzyme.  Derivative 2 had a broader, more
symmetrical protein distribution, the center of which migrated
about 16% faster than unmodified polymerase.  This qualitative
effect was expected, since each conjugated dye molecule contrib-
utes at least one negative charge in the form of a carboxyl group

IIIIIIII
             B

13

-

E,

306

02

240 280 320 360 240 280 320 363 400

Wavelength (mA)

!.5

P

13 ;

3

3.1

FIG. 5. Absorption spectra of DNA polymerase allowed to
react with NCMIA. A, Derivative 2; B, Derivative 4. The
preparation of both derivatives is described in Table 11.  The
solid lines are the absorption spectra of the dye-protein con-
jugate.  A*m is the absorbance measured with a 1-cm light path.
The curve8 formed from individual points are difference spectra
for the conjugated dye obtained by pointwise subtraction of the
unmodified polymerase spectrum from that of each conjugate.
All spectra were normalized to a protein concentration of 1.09
mg ml-' (1W6 M) by first estimating the conjugation stoichiometry
from the absorbances at 280 and 260 mp, applying a correction
factor to   for the dye contribution (less than 3%), and ad-
justing the spectra to a corrected Asso of 0.926 (actual absorb-
ances were within 14y0 of this value).  Protein concentrations
measured by the method of Lowry et al. (12) gave values within
experimental error of those determined spectrophotometrically.
The data were recorded on a Zeiss PMQ I1 spectrophotometer
with 1-cm light path cuvettes at a temperature of 2-10',  Correc-
tions for light scattering were not applied.

523 360 400 440 480

I:IIIIIII
              B

Wavelength (my)

FIG. 6. Fluorescence emission spectra (A) and excitation
spectra (B) of DNA polymerase allowed to react with NCMIA.
The numbers I and 4 refer to the corresponding derivatives of
Table I1 and the number 0 to unreacted polymerase.  Excitation
for the emission spectra was at 290 mp.  In the case of the excita-
tionspectra, Corning CS 3-73 filters were used to isolate the
emission.

and, if conjugated with an amino group of lysine, results in the
removal of a fractional positive charge.  We calculated that a
DNA polymerase molecule has 50 to 70 negative charges under
the conditions of electrophoresis (pH 9.8 at O"), based on its
amino acid composition, the assumption that the ammonia is
derived from amide groups, and the reported values for the in-
trinsic dissociation constants of the amino acid side groups (13).
An incxease of mobility of 16% for Derivative 2 would therefore
correspond to a change of about 8 to 11 negative charges as
compared with the conjugation stoichiometry of 6.

Fluorescence emission spectra of Derivatives 2 and 4 are
shown in Fig. 6A; corresponding data for the other derivatives
are given in Table 11.  Native DNA polymerase had a trypto-
phan emission at 335 mp when excited at 290 mp.  Reaction
with NCMIA reduced the 335 mp emission, and a new peak
appeared at 427 mp.  The effect was more pronounced in the
case of the more highly conjugated derivatives.  The emission
spectrum of the conjugated dye, excited directly at 365 mp, also
had a peak at 427 mp and thus resembled the spectrum of the
presumed amide derivative of NCMIA (Fig. 3).  Together with
the similarity in the absorption spectra, this finding suggests
that a large fraction of the dye may have been conjugated with
amino groups on the protein.  If so, the probable structure would
be the N-crtrboxyanthranilamide (11), with R the side group of
lysine.  The single nonessential sulfhydryl group and NH2-
terminal methionine (I), if involved in the reaction with NCMIA,
cannot account stoichiometrically for the extent of reaction.

The excitation spectra of Derivatives '2 and 4 (Fig. 6B) con-
firmed that the tryptophan emission was quenched because of
energy transfer to the dye chromophore.  The three peaks in the
spectra were in good correspondence with the absorption peaks
(Fig. 5).  The efficiency of energy transfer, T, was obtained

3014

Enzymatic Synthesis of DNA. XXVII

Vol. 244, No. 11

from the escitakion spectra by the following relation (6)

      T = n' (ESO/E334 - cd4 c~~~/&
where E280 and EW are the magnitudes of the excitation spectra
at 280 and 334 mp, respectively; 6280 and 334 are the molar extinc-
tion coefficients of the conjugated dye at 280 and 334 mp with
values of 360 and 3480 M? cm-1 (Fig. 3), respectively;   is the
molar extinction coefficient of the protein at 280 mp and is equal
to 9.26 x 104 M-' cm-'; and n is the number of dye molecules
per protein molecule. The calculated efficiencies of energy
transfer ranged from 20 to 47% (Table 11).  Since these values
were necessarily averages for mixed populations of different
molecules, quantitative deductions as to the probable situation
of dye molecules relative to the nine tryptophan residues in the
enzyme are not justified.

The polarization of the fluorescence emission of the conjugated
dye was measured; values are given in Table I1 in the form of
emission anisotropies.  The excited state lifetime of the con-
jugated chromophore is not yet known; therefore an evaluation
of the rotational relaxation time was not attempted.

The quantum yield of the conjugated dye was not measured
directly; estimated values of 0.4 to 0.6 were obtained by an
indirect method (Table 11).
Enzymatic Proper'ies of NCMIA-DNA Polymerase Conjugates
-NCMIA had pronounced effects upon the enzymatic proper-
ties of DNA polymerase, most notably a marked depression of
polymerase activity (Table 11).  The specific activity of Deriva-
tive 1, having about 6.6 dye molecules per protein molecule, was
reduced by more than 99% (poly d(A-TI-primed assay).  Deriv-
ative 3, with only about 2.3 dye molecules per protein molecule,
showed 10% residual activity.  When measured in the DNA-
primed polymerase assay, the apparent reduction of activity
was less (Table 11).  The derivatives formed in the presence of
dATP retained somewhat more polymerase activity despite only
a slight reduction in conjugation! The presence of Mg++,
which has been found recently to facilitate binding of deoxy-
nucleoside triphosphates (14), did not make the conjugation less
pronounced in preliminary experiments.

The effects on exonuclease activity produced by reaction with
NCMIA were strikingly different from those on polymerase
activity. Low levels of conjugation (Derivatives 3 and 4,
Table 11) actually led to an increase in specific activity of 10 to
20%, whereas higher degrees of reaction (Derivatives 1 and 2)
depressed the activity but only by 20 to 50%.
The relatively greater destruction of the polymerase activity
compared to exonuclease activity suggested the possibility that
a modification of the enzyme in the site for triphosphate
binding and reactions might be responsible for this effect.  To
test this possibility, an NCMIA derivative of the enzyme was
prepared in which polymerase activity was reduced to 0.02%
in a poly d(A-T)-primed assay (Table 111).  The exonuclease
activity was 20% of that of the unmodified enzyme when tested
with a poly d(A-T) substrate under the usual conditions of
assay but 58% at pH 6.5 and 920% with a DNA substrate at
pH 7.4. With this derivative, the functions involving triphos-

3 In another experiment, performed in parallel with that of
Table 111, an NCMIA derivative of the enzyme was formed in the
presence of a saturating concentration of poly d(A-T) oligomer
(8). This enzyme had 25 times more polymerase activity and 3
times more exonuclease activity than enzyme modified in the
absence of poly d(A-T) oligomer. The conjugation stoichiometry
of this derivative was not measured.

phates, such as PP, exchange, stimulation of exonuclease
activity, and binding, were either markedly depressed or unde-
tectable. However, pyrophosphorolysis, in which triphosphates
appear as products, was catalyzed at the relatively high level of
2%.

If the triphosphate site was altered in the reaction with
NCMIA, an increase in K, for triphosphate in the synthetic
reaction might be anticipated.  In order to differentiate between
the rates of phosphodiester bond formation and net synthesis,
simultaneous measurements of both nucleotide incorporation
into product and PPi release were made as a function of triphos-
phate concentration (Fig. 7).  The unmodified enzyme showed
a typical Michaelis-Menten substrate saturation curve with the
same kinetic parameters for the two functions (K, of 8 PM and
vmax of 24,000 units mg-1).  The NCMIA derivative showed a
strikingly different behavior, with a clear dissociation between
synthesis and PPi release.  Whereas synthesis remained at a
constant and low level, the rate of PPi release increased linearly
with substrate concentration, suggesting that the K, for triphos-
phates was in fact much greater.  These results are all consistent
with a profound distortion of the triphosphate site in the
modified enzyme.

In another experiment, the specific activity of synthesis was
shown to be independent of enzyme concentration over a 25-fold
range and was equivalent to the incorporation of only about two
nucleotide units at the end of the polynucleotide chain bound to
a given polymerase molecule.  This calculation assumes the
existence of a uniform population of enzyme molecules.  An
alternative possibility is that all the residual synthetic activity
resided in a small fraction of relatively unaltered enzyme and
that the PP, release represented a catalytic function aborted
before actual phosphodiester bond formation.  The postulate of
a limited synthesis by most or all molecules, however, is consis-
tent with the high level of residual nuclease activity.  It would
appear that the rate of synthesis is so reduced that the product
is rapidly degraded leaving only a small steady state amount at
the end of the chain.  There is effectively, therefore, a turnover
of substrate without a net prolongation.

Assuming the validity of the proposed model, the linear
dependence of PPi release on substrate concentration in the case
of the modified enzyme would be consistent with a much in-
creased K, and thus conditions of limiting substrate for which
u = S.u,,/K,.  The slope of 2.6 X 10' therefore represents
the ratio of the altered kinetic parameters, um.,/K,.  If pyro-
phosphorolysis is the reversal of phosphodiester bond formation,
then thermodynamic considerations stipulate that the ratio of
these activities should not change in the modified enzyme when
measured at equilibrium or even under the nonequilibrium assay
conditions, assuming additionally that the catalytic mechanisms
are not altered, and that the assay for the reverse reaction con-
forms to the same limiting rate law, as stated above.  Although
these conditions have not been established, it is instructive to
consider their possible validity in the light of the experimental
results. The ratio of the specific activities for synthesis (or PPi
release) and pyrophosphorolysis for the unmodified enzyme is
14.9 from Table 111.  The modified enzyme showed 24 units
mg-1 for pyrophosphorolysis, which would correspond to a u,,,
of 360 units mg-l for PPi release assuming the invariance of the
ratio of activities.  It follows from the observed value of vmax/Km
that K, should be on the order of 2 mM.  In other experiments,

Issue of June 10, 1969

T. M. Jowin, P. T. Enylund, and A. Kornberg

3015

18,000b
4,800

450
1,500
2,680
4,740
6,640
6,370
5,460
3,G00
5,020

  13
58
1 , 210
6,050

-5,400

                 TABLE 111
Altered enzymatic activities of an NCMIA derivative of polymerase

The NCMIA derivative was prepared in a reaction mixture  phosphorolysis and PP, exchange were measured according to the
containing 22 ALM enzyme, 30 PM mHg(II), and 1.6 mM NCMIA,  method of Deutscher and Kornberg (X), except that the PPI
in 0.1 M potassium phosphate, pH 7.4.  After 25 hours at 0" the   concentration was 1.3 mM.  PPI exchange rates were calculated
solution was extensively dialyzed against the same buffer.  The  assuming that, for the unmodified enzyme, pyrophosphorolysis is
isolated material had a protein concentration of 1.93 mg ml-l both  50% inhibited by triphosphates under the conditions used (15)
by the method of Lowry et al., and by corrected spectral deter-  but that for the modified enzyme the activities are additive.
minations.  The absorbance ratio, A~so:A2oo, was 0.794 and the  The binding of dTTP to the enzyme was measured by equilibrium
number of conjugated dye molecules per protein molecule was  dialysis at 5" in 0.05 M potassium phosphate, pH 7.4-7 mM MgCIL;
calculated to be 10.7 by the method described in Table 11.  The  the method will be described elsewhere (14). For primer or
mercury was present in the incubation in order to provide a  substrate, 'H-DNA from E. coli was prepared according to the
convenient determinant of concentration.  It had no apparent  procedure of Deutscher and Kornberg (15); the specific activity
effect on the course of the reaction or on the properties of the  was 1,140 cpm nmole-I.  Single strand breaks were introduced
derivative, although this point was not systematically in- by incubation with pancreatic DNase (Worthington) at a level of
vestigated.  All assays were performed at 37" for 30 min under  0.1 pg per pmole of DNA for 100 min at 37" before addition of
conditions for which activity was proportional to enzyme con-  polymerase and triphosphates.  In the case of the poly d(A-T) as
centration.  One unit of activity corresponds to the incorpora-  primer, the triphosphates dATP and dTTPwere addedat the indi-
tion, release, or exchange of 10 nmoles of nucleotide per 30 min.  cated concentrations.  With DNA as primer or substrate, dGTP
The polymerase and nuclease assays were carried out as pre-  and dCTP were also supplied.  Except where indicated, pH

3.2

12

260
260
320
360

1,170
1,410
1,440

940
990
120
120

24
25

-1

viously described (l), except for the indicated changes.

Activity

Polymerase

Exonuclease

Pyrophosp horolysis
Pyrophosphorolysis + PPi
exchange
PPi exchange

Binding of dTTP

Primer or substrate

Poly d(A-T)

DNA (activated, calf thy-

mus)

Poly d(A-T)

DNA (activated E. co2i)

Poly d(A-T)
Poly d(A-T)

Poly d(A-T)

rro- values were at 2.5'.

Tri-
bosphate

PY

33
33

2

333d

33

33

Pi refers to potassium phosphate, Tris to Tris-HCI, and Gly
to sodium glycinate.  The concentration in all assays was 67 mM.
* Exonuclease 111 was not required with this particular poly
d(A-T) primer for the full expression of polymerase activity (1).

saturation of the PPi release function was not achieved at a 0.8
mM triphosphate concentration, a result consistent with the
predicted K,.
Exteni of Conjagation of Enzpne-We wished to determine the
minimum number of dye molecules required for alterations of
enzymatic activity and to assess possible specificity for sites on
the protein.  The maximum stoichiometry of conjugation ob-
served was about 11 dye molecules per protein molecule (Table
111).  One simple model postulates the existence of N equally
reactive sites, of which m are important for enzymatic function,
so that reaction at any one or more virtually abolishes activity.
The fraction, f, of residual activity in a population with an aver-
age of p dye molecules per protein molecule would then be given

7.4
7.4

6.5@
6.98'
7.325-
7. 90c
7. 6gC
8.16O
8.%=
8.65~
8.76c~
7.4
7.4
7.4
7.4

7.4

7.4

Buffer'

Pi
Pi

Pi
Pi
Pi
Pi
Tris
Tris
GlY
GlY
G~Y
Pi
Pi
Pi
Pi

Pi

Pi

I Modified enzyme

Residual
activity

%
0.018
0.25

58
17
12
8
18
22
26
26
20
920
210

2.0

0.41

-0.018

< 10

c pH at 37".
d The activation by triphosphates of the exonuclease function
the unmodified enzyme is described by Lehman (16).

by (1 - p/N)".  Assuming N = 11, then a p of 2.3 andf of 0.1
(Table 11) lead to a value for m of about 10.  The data for Deriv-
ative 1 of Table I1 yield a value form of 6.

KimtiCs of Conjugation of Enzyme-This model was evaluated
further by studying the kinetics of reaction with DNA polym-
erase at several concentrations of NCMIA (Fig. 8).  Depres-
sion of polymerase function proceeded from zero time with
neither a lag nor a transient activation (Fig. 8A).  This finding
is consistent with "one-hit" kinetics, an effect dependent on the
reaction of a single NCMIA molecule.

The time course of exonuclease activity (Fig. 8B) was quite
different from that of the polymerase.  At the highest NCMIA
concentration, the activity at the first time point (0.4 hour) was

3016 Enzymatic Synthesis of DNA. XXVII Vol. 244, No. 11

U /-UnrnodiF,hd enzyme
.e, 20,000
>r

sv)
3t

<5 10,000

.-

+

8

u
0.

[jH-dTTP) or [3'P-T-dATP] GYM)

FIG. 7. Dependence of synthesis and PPi release kinetics on
substrate concentration for unmodified and NCMIA-conjugated
enzyme. The derivative described in Table I11 was used. Syn-
thesis and PP, release were measured concurrently in a reaction
1 volume of 0.6 ml containing 67 mM potassium phosphate, pH 7.4,
6.7 mM MgCL, 1 mM mercaptoethanol, 31 nmoles of poly d(A-T),
the indicated concentrations of "-dTTP (specific activity, 3.7 x
10' cpm nmole-1) and yS2P-dATP (specific activity, 7.7 x 10'
cpm nmole-I), and either 4 ng of unmodified enzyme, 1.5 pg of the
modified enzyme, or polymerase diluent alone.  The poly d(A-T)
was incubated for 30 rnin at 37" prior to the addition of triphos-
phates and enzyme with 0.1 unit of exonuclease I11 per nmole of
polynucleotide in order to increase its priming efficiency (1); the
exonuclease I11 was inactivated by heating for 5 min at 65".
After all additions, incubation was resumed for 30 min.  The
degree of incorporation of 3H-dTME' into polymer was measured
by a modification of the procedure of Falaschi, Adler, and Xho-
rana (17). A 0.1-ml aliquot of each incubation mixture was
plated on a 2-cm square of DEAE-cellulose paper and dropped
into 500 ml of 0.3 M ammonium formate (brought to pH 8 with
NITa).  The papers were shaken occasionally for 10 min at room
temperature and drained.  This process was repeated four times,
followed by two washes each with 50 ml of ethanol and ether.
The air dried papers were counted in 5 ml of a standard toluene
scintillation fluid. The blanks were linearly related to the input
of triphosphate, and the proportionality factor was 0.03%.  PPi
release was measured by a combination of adsorption to charcoal
to remove nonutiliaed dATP and precipitation of interfering azP,
from the supernatant by formation of the insoluble triethyl-
ammonium-phosphomolybdate complex according to the method
of Sugino and Miyoshi (18).  To the remainder of the chilled
incubation mixture (0.5 ml) were added 1 ml of 3 mM sodium
pyrophosphate-0.15 N HCl and 6 drops (0.15 ml) of a 20% sus-
pension of Darco G-60.  After 5 rnin at 0" the suspension was
centrifuged and 0.5 ml of the supernatant fluid was placed in a
polyethylene scintillation vial with 0.05 ml of 2 M Tris base and 10
ml of the naphthalene-dioxane scintillation fluid. Blanks at
this step were proportional to substrate input, with a factor of
1.6%.  Controls indicated that the Darco was capable of ad-
sorbing 99.6% of a pure triphosphate.  Thus about 1% of the
Y-SzP-dATP appeared to have been degraded during the incu-
bation at 37".  The 12Pi so produced was precipitated by treat-
ment of an additional 0.8 ml of the Darco supernatant with 0.1
ml of 4 M HClO,, 0.7 ml of 0.08 M ammonium molybdate, and 0.14
ml of 0.8 M triethylammonium-HC1 (pH 5).  The dense, yellow
precipitate was removed by centrifugation and 1.3 ml of the
supernatant containing the 3lPPi were placed in a polyethylene
scintillation vial with 0.2 ml of 2 M Tris base and 10 ml of scintilla-
tion fluid.  This procedure reduced the blank to 0.5% of the
input triphosphate, thus permitting this experiment with the
modified enzyme when the maximal utilization of substrate was
only 0.7%.

The data for the unmodified enzyme were analyzed by plotting
the observed incorporation, AS, against -In (1 - AS/S,), where
So is the initial concentration of each triphosphate.  This inte-
grated form of the rate equation was used in order to correct for

A Polymerase NCMIA

A

0.2

3

I I 1 I i I 1 I

20 40 60 80

0

Tima in houi-5

FIG. 8. Kinetics of NCMIA reaction with DNA polymerase.
A, Polymerase activity (poly d(A-T)-primed); B, exonuclease
activity.  Incubation waa at 0" in volumes of 12 pl containing
0.98 mg of polymerase ml-1 in 0.1 M potassium phosphate buffer,
pH 7.1, and the indicated concentrations of NCMIA.  The values
are relative to unmodified polymerase (set at 1.0). Aliquots of
2 pl were removed at the indicated times for assay.

actually above the control level and remained so even at 5.5
hours, when 84% of the polymerase activity had been lost.
Only uFon further reaction was a decrease in exonuclease function
observed. The limiting value of 48% to which exonuclease
activity decreased agreed within experimental error with the
values for Derivatives 1 and 2 (Table 11).  At the intermediate
NCMIA concentration, only enhancement of exonuclease activity
was observed and the time course was also compatible with a
single hit process.  The maximum observed increase was about
30% (at 24 hours), with some indication of an incipient decline
at about 72 hours.

In order to determine rate constants, the logarithm of the

the 1 to 8% utilization of substrate in the 30-min incubation
period. A straight line was obtained for both PPI release and
synthesis, yielding a K,,, of 8.1 p~ and a urn.= in the form of a specific
activity of 24,000 units mg-1. The rate corresponding to the
usual substrate concentration of 33 p~ was 19,300, within experi-
mental error of usually observed values.

Issue of June 10, 1969

T. M. Jovin, P. T. Englund, and A. Kornberg

3017

residual polymerase activity in Fig. 8 was plotted against a time
axis corrected for NCMIA hydrolysis.  Reasonably straight
lines were obtained; they yielded an apparent second order rate
constant of 5 M? min-1.  Assuming a value for m of 10, the rate
constant expressed with respect to sites instead of molecules
would be 10 times less, or 0.5 M-1 min-1.  The limited data do
not permit a more detailed analysis, but it is of interest that the
apparent second order rate constant for the reaction of NCMIA
with NHa (20", 13.3 M NHa) was 3.6 x 10-4 b1-1 min-', or three
orders of magnitude less.4

The incubation mixtures of Fig. 8 were analyzed electropho-
retically 2 weeks after the last indicated time point.  The three
proteins, in order of increasing initial NCMIA concentration,
migrated faster than unmodified polymerase by factors of 3%,
5%, and 23%.  By comparison with the data for Derivative 2
(Table 11), it was calculated that the conjugates (Fig. 8) formed
at 0.1 and 1 m~ NCMIA, respectively, had an average of
about 2 and 8 dye molecules per polymerase molecule.  Thus the
initial increase in exonuclease activity observed in Fig. 8B
occurred under conditions where on the average less than one
NCMIA molecule had reacted and suggests that attachment of
a single molecule affects both polymerase and exonuclease activi-
ties concurrently.  From the data of Fig. 8 it was estimated that
the quantitative effect of a single substitution is to increase the
nuclease function of the enzyme by a factor of about 2. Subse-
quent reaction reduces activity, and thus in a mixed population
the full potentiation is not seen.

DISCUSSION

The reactions of DNA polymerase with Hg(I1) confirm the
observation from alkylation experiments that the single sulfhy-
dryl group of the enzyme is not required for activity (1).  The
formation of a polymerase dimer bridged by a mercury atom
indicates that the cysteine side chains are located in a relatively
exposed site on the surface of the molecule.  It is of interest that
both members of the dimer retain full enzymatic activity.  The
possibility of forming dimeric structures through a direct disul-
fide bond, as is possible with serum albumin (19), was not ex-
plored.  If such dimers or related forms exist in the cell, they
would not have been detected because of the use of reducing
agents in our purification procedure.

DNA polymerase exhibits a quite different behavior in its
reaction with the fluorescent acylating agent, N-carboxymethyl-
isatoic anhydride.  A product of limited acylation containing an
average of 2.3 moles of dye per mole of protein showed a 20%
increase in exonuclease, but its polymerase activity was reduced
by 90%.  In a more extensive reaction in which approximately
11 dye molecules were conjugated to the enzyme, only 0.02% of
the polymerase activity remained while 20% or more of the
exonuclease activity was preserved with poly d(A-T) as a primer
or substrate.  These are the first instances of selective inactiva-
tion of the polymerase function, excluding the effects seen after
treatment with guanidine-HCl and mercaptoethanol and de-
scribed in the preceding paper (1).  Prior studies have shown
the constancy of the polymerase to nuclease ratio throughout
purification and during heat and urea inactivation (20).  These

4 This calculation does not take into account the probable
nature of the reactive species in concentrated ammonia solutions.
In reactions of isatoic anhydride and N-methylisatoic anhydride
with n-butylamine at 1.3", rate constants of 180 M? sec-1 and
86 ~-1 sec-1, respectively, have been reported (9).

and related observations (8, 14, 15) suggest, therefore, that the
two catalytic functions are carried out in the same general active
center of the molecule but that the sites do not correspond
exactly.
Inactivation of an enzyme by a modifying agent may often
involve an alteration rather than abolition of function, as through
shifts in pH maxima and relative affinities to substrates and
cofactors.  This point is supported by studies relating the vari-
ous activities of polymerization, hydrolysis, pyrophosphate
exchange, and pyrophosphorolysis.  The finding of approxi-
mately 11 reactive sites is of interest because there are potentially
a much greater number of suitable groups on the protein, e.g. 61
c-amino groups of lysine.  It is possible that sites of unusual
reactivity are involved: in this regard the much lower reaction
rate of NCMIA with ammonia is noteworthy.

The functional properties of the modified enzyme and direct
binding measurements indicate a large reduction in the affinity
of the enzyme for the deoxyribonucleoside triphosphates.  How-
ever, other sites and effects must be involved.  The protection
from inactivation by poly d(A-T) suggests some capacity to
redirect the course of reaction but probably not through direct
interaction with NCMIA, since isatoic anhydride has been
reported to be nonreactive toward phosphate and phosphate
esters, including nucleotides (21).  It will be of interest to deter-
mine the effect of structure of various nucleic acids and nucleo-
tides on this protective function.

The altered pattern of nuclease function also indicates involve-
ment of sites other than that for the triphosphates.  Shifts
in the pH spectrum of activity were observed, along with indica-
tions of altered influences of ionic strength.  More remarkable
was the fact that at pH 7.4 the extensively conjugated enzyme
was almost 10 times more effective with a DNA substrate but
10 times less effective with poly d(A-T) as compared with the
original exonuclease, although in terms of absolute rates the
poly d(A-T) was still the better substrate.  Such an effect may
reflect subtle changes in the influence of polynucleotide secondary
structure upon the interaction with the enzyme.

To what extent the exonuclease measurements reflect the
act.ivity on the 3' terminus as compared with the activity directed
toward the 5' terminus (22) has not been determined.  In view
of the recent evidence that the regions in the active center of the
enzyme responsible for these two exonuclease activities of DNA
polymerase are distinctly different,' it becomes of great interest
to analyze the modified enzyme for each of these activities.

The kinetic model proposed for the inactivation of the enzyme
is not unique, owing to the probable existence of sites with vary-
ing reactivity and functional importance.  Clarification, of this
problem requires the preparation and isolation, as by ion ex-
change chromatography, of molecules with known and homoge-
neous extents of conjugation.  In order to refine the spectral
studies, additional information about NCMIA and its derivatives
is required.  Model compounds would be of help in the evalua-
tion of the nature of the substitution and the polarity of the
modified site.  Better quantitative determination of conjugation
stoichiometry would follow from the use of radioactive reagent.
It remains to be seen whether energy transfer, quantum yield,
and polarization measurements are sensitive to the interaction
of the enzyme with DNA and substrates.  Finally, similar

6 N. R. Cozzarelli, R. B. Kelly, and A. Kornberg, manuscript

in preparation.

3018 Enzymatic Synthesis of DNA. XXVII Vol. 244, No. 11

studiea with other polymerases and reagents should indicate
whether the phenomenon is a general one or specific to NCMIA
and the E. coli enzyme.

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