THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 262, No.

1, Issue of January 5, gp: 47&485,1987

0 1987 hy The American Society of Biological Chemists, Inc. rrnted in U.S.A.

Action Mechanism of Escherichia coli DNA Photolyase

I. FORMATION OF THE ENZYME-SUBSTRATECOMPLEX*

(Received for publication, June 11, 1986)

Gwendolyn B. Sancar, Frances W. Smith, Ralph Reid, Gillian Payne, Michael Levy, and AzizSancar
From the Department of Biochemistry 231H, Universityof North Carolina Schoolof Medicine,
Chapel Hill,North Carolina 27514

Escherichia coli DNA photolyase (photoreactivating Escherichia coli (Rupert et al., 1958) and the action of the
enzyme) is a flavoprotein. The enzyme binds to DNA enzyme has been studied extensively in vivo (Harm et al.,
containing pyrimidine dimers in a light-independent 1968; Harm, 1970a, 1970b), until recently the paucity of the
step and, upon illumination with 300-600 nm radia- enzyme (about 20 molecules/cell in wild type cells) had made
tion, catalyzes the photosensitized cleavage of the cy- in vitro studies of the enzyme in a fully defined system
clobutane ring thus restoring the integrity of the DNA. impossible. Several years ago we began a concentrated effort
We have studied the binding reaction using the tech- to obtain sufficient protein to undertake studies on the mo-
niques of nitrocellulose filter binding and flash photol- lecular mechanism of action of photolyase; to that end we
ysis. The enzyme binds to dimer-containing DNA with cloned the phrgene (Sancar and Rupert, 1978b), constructed
an association rate constant kl estimated by two dif- overproducing E. coli strains in which >15% of total cellular
ferent methods to be 1.4 % 10' to 4.2 x 10' M-' s-'. The
protein is photolyase, and purified the enzyme to homogeneity
dissociation of the enzyme from dimer-containing DNA
displays biphasic kinetics; for the rapidly dissociating (A. Sancar et al., 1984b). Pure photolyase consists of an M,-
class of complexes = 2-3 % s-l, while for the 53,994 polypeptide (G. Sancar et al., 1984) and two intrinsic
more slowly dissociating class k2 = 1.3 X to 6 X chromophores, a blue neutral FAD radical and a second
s-'. The equilibrium association constant Ka,as chromophore the chemical identity of which is not yet known
determined by the nitrocellulose filter binding assay (Sancar and Sancar, 1984; Jorns et al., 1984); in vitro the
and the flash photolysis assay, was4.7 % lo7to 6 X lo7 enzyme is capable of repairing pyrimidine dimersin the
M", in reasonable agreement with the values predicted absence of any additional proteins or cofactors and at a rate
from kland kz.From the dependence of the association similar to that observed in vivo. Using this fully defined
constant on ionic strength we conclude that the enzyme system we report in this and the following two papers the
contacts no more than two phosphodiester bonds upon results of studies on the thermodynamic, kinetic, and photo-
binding; this strongly suggests that the pyrimidine di- chemical processes involved in photoreactivation by the E.
mer is the main structural determinant of specific pho- coli enzyme. In this article we deal with the first half of the
tolyase-DNA interaction and that nonspecific ionic in- reaction sequence, formation of the enzyme-substrate com-
teractions do not contribute significantly to substrate plex.
binding. We have previously reported that purified photolyase binds
specifically to UV-irradiated DNA and that this binding is
relatively insensitive to changes in salt concentration or pH
(G. Sancar et al., 1985). In thisreport we extend those studies
Irradiation of DNA with 254 nm of light induces the for- by quantitating the specific association constant K A using
mation of dimers between adjacent pyrimidine bases in the both the nitrocellulose filter binding assay andthe flash
same DNA strand; leftunrepairedsuch lesions resultin photolysis-transformation technique; in addition we have
mutation or cell death. DNA photolyases (EC 4.1.99.3) are a quantitated the kinetic constants for complex formation, kl
group of enzymes which catalyze the light-dependent reversal and k2. Both equilibrium and kinetic methods yield estimates
of pyrimidine dimers in DNA thus repairing these lesions in for KAof 4.7 X lo7to 1.4 X 10' M-', values which are unusually
situ. Thereaction proceeds by a two-stepmechanism (Rupert, low for a site-specific DNA-binding protein. In addition,
1962): (i) the substrate binding step, which can occur in the quantitation of the effect of NaCl concentration on KA indi-
dark, and (ii)dimer reversal, which occurs only in the presence cates that the enzyme contacts only 1-2 phosphates on the
of light, the precise wavelength being dependent upon the DNA backbone when bound to the pyrimidine dimer; taken
source of the enzyme (see Harm, 1976, for a review). Thus together withthe relatively low k1 (4.2 X lo6 M" s-') exhibited
the overall reaction scheme can be written as the following. by the enzyme, these results suggest that in contrast to other
k, k3
site-specific DNA-binding proteins, nonspecific ionic inter-
E + S " E S " E + P actions contribute very little to the equilibrium or kinetics of
k2 hv photolyase binding.
Although the first evidence for the enzymatic nature of
MATERIALSANDMETHODS
photoreactivation was obtained using crude extracts from
Enzyme Preparation!-E. coli DNA photolyase was purified to
* This work was supported by National Institutes of Health Grant homogeneity as described previously (A. Sancar et al., 1984b) with
GM32833, National Science Foundation Grant PCM 51212, and a the modification that at Step VI, Blue Sepharose (Sigma) was sub-
grant from the Miller High Life Foundation. The costs of publication stituted for DNA cellulose; this change enabled us to obtain pure
of this article were defrayed in partby the payment of page charges. enzyme at higher yield (about 10% versus 5.4%) than previously. The
This article must therefore be hereby marked "advertisement" in enzyme had a blue-purple colorwhen freshly prepared due to its
accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. neutral FAD radical cofactor (Jorns et al., 1984) and concentrated

478
 Binding of Photolyase to DNA 479
solutions (5-10 mg/ml) retained this color for over a year when stored the flash. (See also Jorns etal., 1986, in this series.) Originally Rupert
a t -80 “C. We willrefer to such enzyme as “blue enzyme.” In contrast and colleagues used Yashika PRO 50 photographic flash units to
to the stability of the enzyme under the conditions described above, provide the flash. We have found that any of several commercially
prolonged purification procedures and/or storage a t -20 “C in 40% available photographic flash units are equally applicable provided
ethylene glycol containing 10 mM 0-mercaptoethanol for more than that thesample is screened with Pyrex glass and/or the plastic lid of
6 months results in the appearance of “yellow enzyme.” Spectropho- a Petri dish to eliminate short wavelength radiation. In the studies
tometric analysis (absorption a t 450 and 580 nm) indicated that 70% described in this paper we used Vivitar 2500 photographic flash units
of the FAD in yellow enzyme was in the fully oxidized form (Jorns et for flash photoreactivation.
al., 1987). The oxidized FAD remained firmly associated with the Substrate for the flash photolysis assay was UV-irradiated pBR322
enzyme as overnight dialysis of the yellow enzyme did not result in a DNA, labeled and irradiatedas described above.Following flash
detectable decrease in the 450-nm absorption. photoreactivation, photolyase was removedby phenol extraction and
Visualization of ES Complexes by Electron Microscopy-DNA mol- the DNA was removed from photolyase assay buffer and concentrated
ecules that contained pyrimidine dimers in a slightly off-center loca- by ethanol precipitation. An aliquot of the DNA wasthen used in the
tion were prepared as follows. Plasmid pBR322 DNA was digested transformation assay to determine the number of UV-induced lesions
with EcoRI and BanHI restriction endonucleases and the (EcoRI- and pyrimidine dimers remaining per molecule. Losses during extrac-
B U ~ H I )and ) ~ ~ , B were separated on aga- tion and precipitation were monitored by scintillation counting.
~ , (~B ~ ~ H I - E C O R Ifragments
rose gels and purified as described by Vogelstein and Gillespie (1979).
The small fragment was irradiated with 1.5 kJ/m2 of 254 nm of light RESULTS
to produce an average of 3 pyrimidine dimers/molecule. The irradi-
ated DNA was mixed with the nonirradiated ( B ~ ~ H I - E C O R I ) ~ ~ ~ Visualization of Photolyase-DNA Complexes
fragment a t a concentration of 4 pg/ml each and covalently linked
using T4 DNA ligase. After overnight incubation a t 14 “C, ligase was We have previously reported (G.Sancar et al., 1985) that
inactivated by heating at 65 “C for 10 min. The DNA was then photolyase binds specifically to UV-irradiated DNA and has
digested with PuuII (which cuts a t base pair 2067) and separated on little affinity for nonirradiated DNA. This is also apparent
a 1 % agarose gel; a full-length fragment (4363base pairs) was purified
from this gel and used for electron microscopic studies. Photolyase- from analysis of DNA/photolyase mixtures by electron mi-
DNA complexes were fixed with 1%formaldehyde, 0.6% glutaralde- croscopy. Plasmid pBR322 DNA containing dimers only in a
hyde, mounted onto thin carbon films, and rotary shadowed with
tungsten (Griffith and Christiansen, 1978); micrographs were taken
using a Phillips EM400 TLG electron microscope.
Preparation of Substrate for Filter Binding and Flush Photolysis
Studies-Radiolabeled pBR322 DNA was obtained from E. coli
AB2463 (recA13)/pBR322 as previously described (G. Sancar et al.,
1985). The specific activity was 1.3 X lo5 cpm/pg. The DNA was
diluted to a concentration of 20 pg/ml and irradiated with 254 nm of
light. The average number of UV-induced lethal lesions/molecule was
determined by transformation selecting for tetracycline resistance
(Sancar and Rupert, 1978a; A. Sancar etal., 1984a, 1984b; G . Sancar
et al., 1985); incubation of UV-irradiated DNA with a molar excess
of photolyase for 1 h under photoreactivating light (supplied by a
Sylvania F15TF8BLB bulb a t a fluence rate of approximately 10
J/m2), followed by transformation was used to quantitate the average
number of pyrimidine dimers/molecule. When performed as de-
scribed, the transformation assay is a sensitive and accurate method
for quantitating dimer repair. Typically transformation with 0.1 pg
of plasmid DNA containing an average of 6 pyrimidine dimers/
molecule results in 500 (&lo%)transformants/ml of culture, while
cultures transformedwith plasmid molecules containing fewer dimers
contain proportionately larger numbers of transformants according
to the relationship S/S, = e-A,where X is the number of pyrimidine
dimers/molecule.
For experiments to determine k2, competing DNA was prepared by
irradiating plasmid pBM150 (tetnamp’; Johnston and Davis, 1984)
a t a concentrationof 20 pg/ml with sufficient 254-nm light to produce
100 dimers/plasmid molecule. Plasmid DNA was concentrated by
ethanol precipitation and the concentration was determined by ab-
sorbance a t 260 nm.
Nitrocellulose Filter Binding Assay-The details of this assay have
been published previously (Riggs et al., 1968; Madden et al., 1973;
Seawell et al., 1980; G. Sancar et al., 1985). Briefly, the reaction
mixture (110 pl) consisted of 50 mM Tris, pH 7.5, 1 mM EDTA, 10
mM 8-mercaptoethanol, 100 pg/ml bovine serum albumin, DNA a t
the concentrations indicated in the figure legends, and 100 mM NaCl
unless stated otherwise. After addition of photolyase to the desired
concentration, the reaction mixture was incubated a t 22 “C for 1 h
before filtering through Schleicher and Schull BA85 filters (24-mm
diameter). All experiments were performed under illumination from
G.E. gold fluorescent bulbs. FIG. 1. Binding of photolyase to linearized pBR322 DNA
Flush Photolysis-This method is amodification of Porter’s (1960) containing pyrimidine dimers in a slightly off-center location.
classic flash photolysis technique adapted by Harm andRupert (1968) The pBR322 substrate, prepared as described under “Materials and
to thestudy of the action mechanism of photolyase. DNA-photolyase Methods,” was incubated with photolyase a t a concentration of 1.8
complexes presentina reaction mixture are subjected to a high nM DNA and 9.3 nM enzyme under standard binding conditions for
intensity flash of photoreactivating light of approximately I-ms du- 1 h; photolyase-DNA complexes were then fixed and processed for
ration, resulting in repair of pyrimidine dimers bound by the enzyme transmission electron microscopy as previously described (Griffith
at the instantof the flash. Since the turnover rate of the enzyme is and Christiansen, 1978). Bar indicates 100 nm. Upper panel shows
considerably longer than the duration of the flash, the number of one photolyase-DNA complex while the DNA molecule in the lower
dimers repaired (as determined by biological or chemical methods) is panel appearsto bear two complexes;it should be noted that less than
a measure of the number of ES complexes present at themoment of 10% of the molecules analyzed contained two ES complexes.
480 Binding of Phl otolyase to DNA
lyase binding to DNA as a monomer (data not shown). This
observation isin agreement with our previous results obtained
0.8 "h from gel filtration studieswhich showed that photolyase elutes
as a M, = 49,000 protein (A. Sancar et aL, 1984b). Because of
difficulty in visualizing proteins as small as photolyase by
electron microscopy we have not obtaineda sufficient number
of photographstoquantitatethe specific and nonspecific
association constants by this method; however, we may draw
the qualitative conclusion from this and other micrographs
that photolyase does not bind to nonirradiated DNA to any
significant extent.

Quantitation of the Association ConstantKA of Blue

Photolyase
Nitrocellulose Filter Binding-The nitrocellulose filter
binding technique first used to study Lac repressor-operator
PhotolyaseConcentration (nM)
interactions hasbecome a standard techniquefor quantitating
enzyme-substrate complex formation for DNA-binding pro-
FIG.2. Binding of photolyase (blue enzyme) to 'H-labeled teins (Riggs et al., 1968,1970). We haveutilized this technique
UV-irradiated pBR322 DNA. Photolyase at theindicated concen- to study theequilibrium binding of photolyase to UV-irradi-
trations was incubated with 3H-labeled pBR322 DNA containing an
average of 5.5 pyrimidine dimers/molecule and a t a plasmid concen- ated DNA. As can be seen inFig. 2, incubation of radiolabeled
tration of2.34nM. Following 1-h incubation in the dark, duplicate pBR322 DNA containing an average of 5.5 pyrimidine dimers/
samples were filtered through nitrocellulose and the counts retained molecule with increasing concentrationsof photolyase results
were determined by scintillation counting. Symbols: 0, A, and 0, in increasing retention of the DNAby the nitrocellulose filters
values obtained in three independent experiments. The solid line up to the point at which saturation is apparently obtained.
drawn through the data points is the expected retention for KA= 6.0 However, the relationship between the fraction of total DNA
X IO7 M-', while the dashed lines show the expected retention for Ka
= 8.1 X lo7 M" (top) and 3.9 X lo7 M" (bottom).
retained on the filter and the total numberof ES complexes
present in solution is nonlinear in this case because 1) the
1.0 efficiency of retention of molecules containing a single ES
-aa 0.9
60 06
50.05
40.04 - complex is less than 1.0 forphotolyase-pyrimidine dimer
complexes (G.Sancar et al., 1985) and 2) the substrate (pyrim-
z
a 0.8 A';$ 5
12.96 E idine dimers) is not distributed uniformly among the DNA
11.11
0.7 9.26 9 molecules, but rather follows aPoisson distribution. As is
0.6 7.41 g described in the Appendix,' beginning with the relationship
r c
0
0.5
5.56
0
derived by Woodbury and von Hippel (1983) for a substrate
0.4 3.70
0 with multiple independent binding sites,we have derived the
El
VI following equations which express the quantitative relation-
0.3 -2r
0
ship between the fraction of total DNA retainedby the filter
-
c
.-
0
0.2
1.85
0
f (R),the retentionefficiency (t), the average number of pyrim-
e
LL
1.0 idine dimers/molecule (X), the total protein concentration
0 ( P t ) ,the DNA concentration (D), and association
the constant
IO6 10' io9
KA
KA:
FIG. 3. Expected retention (R)by nitrocellulose filters of
UV-irradiated pBR322 DNA as a function of the association
constant (KA) for photolyase-pyrimidine dimer binding. Equa-
tions 1-3 were used to generate curves showing expected retention at where
each of the indicated photolyase concentrations for binding of
pBR322 DNA a t a concentration of 2.34nM and containing 5.5 KAP = KA(P1 - E S ) = CY + ( 2 + KAP,)" (2)
pyrimidine dimers/molecule; a value of 0.34was used for c in Equation
1 (G. Sancar et al., 1985). The data points shown in Fig. 2 were then and
plotted on thesecurves to obtain the KA values. Symbols are thesame
as described in the legend to Fig. 2. CY %[KAP,- (1 + XKAD)]. (3)

KAwas determined from the filter binding data

shown in Fig.
374-base pair region in a slightly off-center location relative 2 as follows: the knownvalues for P,, D, X, and t (=0.34;
to the endsof the linearized molecule was mixed with photo- quantitated by titrating enzyme with dimer-containingDNA,
lyase in the dark and examinedby electron microscopy. Fig. G . Sancar et al., 1985) were used to generate theoretical
curves
1 shows two representatives of the majorityof ES complexes of predictedretention ( R ) versus log KA for eachprotein
we observed. The enzyme binds with measurable frequency concentration (Fig. 3). The experimentally observed retention
only to the irradiated region and binding to thisregion is not at each protein concentrationwas then plotted on the appro-
seen if the mixture isexposed to photoreactivating light prior
to fixing the complexes withglutaraldehyde(notshown). 'The "Appendix" are presented in miniprint at the end of this
From the micrographs shown in Fig. 1 it is appafent that paper. Miniprint is easily read with the aid of a standard magnifying
photolyase is a spherical molecule of about50 A diame- glass. Full size photocopies are available from the Journal of Biolog-
ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request
ter; furthermore, under the same conditions and magnifica- Document No. 86M-1951, cite the authors, and include a check or
tion, DNA polymerase I (M, = 103,000) and UvrA protein money order for $1.60 per set of photocopies. Full size photocopies
( M , = 104,000) appear to be abouttwice the size of photolyase are also included in the microfilm edition of the Journal that is
(Mr = 53,994; G. Sancar et al., 1984) consistent with photo- available from Waverly Press.
 Binding of Photolyase to DNA 481
priate curve and the K A values thus obtained (Fig. 3) when enzyme molecules/dimer and with an association constant K A
averaged gave a value for K A = 6.0 f 2.1 X lo7 M-' for the = 4.7 X lo7 M-' in good agreement with the value for K A
binding of photolyase to pyrimidine dimers inDNA. The good obtained by the nitrocellulose filter binding technique. It
agreement between the observed retention of dimer-contain- should be noted that at great (200-2000-fold) enzyme excess,
ing DNA and that predicted for this K A is shown by the fact only about 75% of dimersare repaired by a single flash
that thesolid curoe drawn through the data points in Fig. 2 is suggesting that some enzyme molecules are able to bind
the theoretical curve calculated from Equations 1-3 using K A pyrimidine dimers but are deficient in photolysis (see below
= 6.0 X lo7 M-', while the dashed lines indicate the retention and G. Sancar et al., 1987, in this series).
expected for K A values of 1standard deviation from the mean.
Flash Photolysis-As an increasing proportion of the sub- Binding of Yellow Photolyase to Pyrimidine Dimers
strate-binding sites on each DNA molecule are bound by E. coli photolyase contains a FAD neutral blue radical
photolyase, the nitrocellulose filter binding assay becomes (Jorns et al., 1984) and has a blue-purple color when freshly
less sensitive to increases in ES complexes/molecule; in ad- prepared. Upon storage at -20 "C the radical is oxidized to
dition, as can be seen in Fig. 3 and Equations 1-3, as satura- FAD,, and the enzyme turns yellow. Since at present we are
tion is approached small differences in the apparent fraction unable to oxidize the blue radical in an experimentally con-
of DNA molecules retained make large differences in the trolled manner, we do not have fully oxidized enzyme. How-
apparent K A . Therefore, we chose to use a second method to ever, we have obtained an enzyme preparation in which 70%
confirm our estimate of K A , namely the flash photolysis of the blue neutral radical has been converted to FAD,, (Jorns
technique originally used by Harm and Rupert (1968, 1970) et al., 1987) after 6 months of storage at -20 "C. When we
to study binding of yeast photolyase in vitro. Various concen- conducted the filter binding experiment with this enzyme
trations of photolyase were mixed with UV-irradiated DNA preparation the results shown in Fig. 5 were obtained. The
and after 1 h of incubationin the dark, the mixture was yellow enzyme preparation was still capable of binding spe-
exposed to a single intense flash of photoreactivating light cifically to UV-irradiated DNA, displaying an apparentasso-
sufficient to repair all dimers complexed with active enzyme; ciation constant K A = 4.8 X lo7 M-'. Clearly the binding seen
determination by transformation assay of the number of is in excess of that expected if only the remaining enzyme
dimers/DNA molecule before and after the flash provides a containing neutral blue radical were capable of binding DNA
direct measure of the number of ES complexes present at the (Fig. 5 , broken line).
time of the flash. As can be seen in Fig. 4,a typical enzyme
saturation plot is thus obtained. Analysis of the data by an Dependence of KA on [NaCl]
Eadie-Scatchard plot (Segel, 1975) is shown in Fig. 4; photo-
When proteins bind to DNA by ionic interactions between
lyase binds to dimers with an apparent stoichiometry of 0.75 positively charged groups on the protein and negative charges
on the DNA phosphate backbone, counter ions are displaced
from the phosphates as the result of binding. Therefore, if an
important fraction of the total binding energy is this ionic
interaction, KA shows astrong dependence on the cation
concentration in the reaction mixture. Record et al. (1976)
have derived a quantitative relationship between K A , ion pairs
formed upon binding, and thecounter ion concentration:

where x is the fraction of counterion [Na] bound per DNA

O6 I

[E SI
[SI,
FIG. 4. Eadie-Scatchard plot of the equilibrium binding of
photolyase to UV-irradiated pBR322 DNA as measured by
the flash photolysis assay. 3H-pBR322 DNA at a concentration of
PhotolyaseConcentration (nM)
3.84 nM and containing an average of5.1 pyrimidine dimers/molecule
was incubated with photolyase at the indicated enzyme concentra- FIG. 5. Binding of photolyase yellow enzyme to UV-irradi-
tions under standard reaction conditions. Formation of ES complexes ated pBR322DNA. Incubation of photolyase with 3H-labeledUV-
was quantitated by the flash photolysis technique followed by the irradiated pBR322 DNA and quantitation of binding by the nitrocel-
transformation assay as described. In this experiment 75% of pyrim- lulose filter binding assay were as described in the legend to Fig. 2
idine dimers (14.7 nM) were repaired by a single flash at saturating except that the DNA concentration was 1.7 nM and contained 6.0
enzyme concentrations; the concentration of ES complexes has been pyrimidine dimers/molecule. The solid line shows binding expected
corrected in the inset and Figure to reflect this fact. The line is a for Ka = 4.8 f 0.57 X lo' M-! obtained from this data, while the
linear least squares fit of the data obtained from the binding isotherm broken line indicates the binding expected if only the 30% of the
(also corrected for repairof 75%of ES complexes with a single flash) enzyme containing the blue neutral radical is active in binding to
shown in the inset. dimers.
482 Binding of Photolyase to DNA
TABLEI determined from the equation
Effect of[NaClI on KAof E . coli photolyase
NaCl concentration K.4
mM
125 5.7 +- 1.7 X lo7 where [ES],is the concentration of complexes immediately
200 5.3 +- 2.1 X 1 0 7
prior to the addition of competitor and [ESIr is the concen-
275 3.3 +- 1.8 X 107 tration of complexes at some later time t after addition of
350 3.8 +- 1.7 X lo7
400 9.5 -I- 4.4 x lo6competitor. If competition is 100% efficient and if all com-
450 7.1 +- 2.5 X lo6 plexes have the same k2 then a semilog plot of In [ E S ] J [ E S ] ,
500 1.2 +- 0.24 X 107uersus t should yield a straight line with a slope of -k2 until
the system nears thenew equilibrium state at which a small
number of ES complexes remain bound to dimers pBR322. on
phosphate (=0.88, Lohman et al.,1980) and m‘ is the number To correct for this lattereffect we have subtracted the fraction
of ion pairs formed between the DNA and protein. Table I of ES complexes remaining at 60 and 120 min (28%) from
shows K A for photolyase binding as a function of [NaCl] as [ES],and [ESIton the assumption that this approximates the
determined by the filter bindingassay. From the slope of the
Record-Lohman plot (Fig. 6) we obtain m‘ = 1.6 f 0.3. These
results indicate that photolyase makes ionic contact with only
1-2 phosphates in the phosphodiester backbone when bound
to thedimer.
Extrapolation of Kobato 1~NaCl concentration can be used
to estimate the nonelectrostatic contribution (AG,,) to the
total free energy of photolyase-DNA binding (Record et al.,
1976). From the data shown in Table I and Fig. 6 we obtain
AGIM= -8.9 kcal/mol a t 20 “C. Since:
AGme = AG,M - m‘ AGys (5)
(where AGlYs= 0.2 kcal/mol, the standard free energy of a
lysine-phosphate ionic interaction at 1 M NaCI), we obtain
AG,, = -9.2 kcal/mol. Under our standard binding conditions
AG = -10.5 kcal/mol and thus only about 10% of the total
free energy of binding of photolyase to a dimer-containing
region is due to ionic interactions.
FIG. 6. Dependence of the association constant K Aon NaCl
Determination of the Rate Constants kl and k2 concentration. The binding constant at each NaCl concentration
was determined as described in the legends to Figs. 2 and 3 except
that the reaction mixture contained 10 mM Tris, pH 7.5, and NaCl
From the reaction scheme for photolyase-substrate binding
at the appropriate concentrations. The error bars indicate the stand-
k1 ard deviation of the mean for each K A determination. The line was
E-kSclES, obtained by linear least squares regression analysis which indicated
k, a correlation coefficientof 0.87. m‘ is the slope and equals the number
of protein-phosphodiesterbond contacts, as described in the text.
the change in concentration of ES complexes in the absence
of photolysis is given by the differential equation

= k , [ E ] [ S ]- k [ E S ] (6)
dt
where [ E ] and [SI are the concentrationsof free enzyme and
. 0.6

free substrate. Becauseof the complexities involved in trying

to directly quantitate [ES]by nitrocellulose filter binding in
our system(see “Determination of K A by Nitrocellulose Filter
Binding,” above) we have utilized the flash photolysis and
transformation techniques to quantitate photolyase-pyrimi-
dine dimer binding.
To determine the dissociation rate constant k2 we utilized
the “cold DNA chase” methodof Riggs et al. (1970) and Harm
and Rupert (1970). Photolyase was incubated with UV-irra-
Time (seconds)
diated pBR322 DNA and incubated for 1 h to allow binding
t o reach equilibrium; competing pBM150 DNA containing a FIG. 7. Determination of k, by the decrease of ES complexes
20-fold molar excess of pyrimidine dimers compared to the in pBR322 in the presence of competing substrate. UV-irradi-
ated pBR322 DNA containing an average of 4.2 dimers/molecule was
pBR322 DNA was then added and at variouslatertimes incubated at a concentration of 3.3 nM with photolyase at 70 nM
samples were removed and exposed t o flash photolysis thus under standard reaction conditions in the dark for 1 h. Competing
repairing dimers in ES complexes at the time of the flash. UV-irradiatedDNA (pBM150 containing approximately 100 dimers/
Quantitation of ES complexes was by the transformation genome) was added at a concentration of 2.66 nM at zero time; at
various later times samples were taken and the number of ES com-
assay selecting for tetracycline-resistant transformants. Ide- plexes remaining were determinedby flash photolysis and transfor-
ally under the reaction conditions describedabove, the term mation assay. kz was determined by extrapolation of the initial slope
k l [ E ] [ S ]in Equation 6 becomes negligible and thuskz can be (15- and 30-9 time points) to a value of 0.37.
 Binding of Photolyase to DNA 483
by adding photolyase to a reaction mixture containing UV-
10.0 -
-
irradiated pBR322 DNA and subjecting the mixture to flash
photolysis at various later times. As can be seen, even using
8.0 - the minimum concentrations of enzyme and substrate which
-I allow us to reliably quantitate [ES] by transformation, the
2 6.0- reaction is very rapid. Only for the 5-s point shown in Fig. 8
'0 - is kl[E],[S], less than 20% of k2[ESIt (using kp = 3 x lo-' s-');
- -
x
using Equation 8 and this first pointwe obtain a value for k,
4.0
I
= 4.2 X lo6 M" s-'. If, on the other hand, we use Equation 9
to determine kl, the values obtained are dependent upon the
2.0 - value chosen for k2 as well as the proportion of the fast and
slowly dissociating complexes present at each time point.
Utilizing Equations 9-11 and kz = 3 x lo-' we obtain a value
5 10 15 20
for kl = 1.4 X lo6 M" s-'.
Time (seconds)
It is of interest to compare the equilibrium association
FIG. 8. Kinetics of formation of photolyase-pyrimidine di- constant K A predicted from the relationship K A = k l / k p with
mer complexes. Reaction mixtures consisted of 0.45 nM pBR322
DNA containing an average of 5.4 pyrimidine dimers/molecule (= the value obtained by the equilibrium binding experiments
2.43 nM pyrimidine dimers) in standard reaction buffer. Reactions described above. Using values for kl of 1.4 x lo6 to 4.2 X lo6
nM; at the indicated M-' s" and for kz of 3 x lo-' s"we calculate K A = 4.7 X lo7
were begun ( t = 0) by addition of photolyase to 8.2
times the reaction mixture was subjected to flash photolysis andthe M" to 1.4 X lo8 M - ~in , good agreement with the values of 4.7
number dimers repaired (and hence the number of ES complexes X IO7to 6 X lo7 obtained by equilibrium binding.
present at the time of the flash) were quantitated by the transfor-
mation assay. DISCUSSION

new equilibrium value (data not shown). The results of a Photolyase is a spherical molecule of about 50 A diameter
typical experiment are shown in Fig. 7 from which it can be which binds specifically to irradiated DNA. Since the enzyme
seen that 1)a large fraction of the complexes dissociate very does not act on photoproducts other than pyrimidine dimers
quickly and 2) the decrease in the fraction of [ES] complexes (Brash et al., 1985) we conclude that this specificity is the
with time is not linear. We interpret this latter resultas result of photolyase-pyrimidine dimer binding. Perhaps the
reflecting the presence of at least 2 classes of [ES] complexes most interesting and surprising result of this study is the
with respect to dissociation rate. The rapidly dissociating relatively minor contribution of electrostatic interactions to
class comprises approximately 65% of ES complexes present the binding of photolyase to substrate. The dependence of K A
at time 0 and (from this and threeother experiments) hasan on monovalent cation concentration indicates that theprotein
estimated k2 = 2-3 X lo-' s-l while the remaining complexes makes electrostatic contactswith only 1-2 phosphates on the
dissociate with an apparent rate constant & = 1.3 X to 6 DNA backbone. This conclusion is consistent with other
X s-'. The presence of two classes of ES complexes which observations: (i) theenzyme can act efficiently on substrates
differ in their kinetic parameters is not unexpected as Harm as small as oligo(dT), (Jorns et al., 1985); (ii) photolyase does
(1970a, 1970b) obtained similar results for the dissociation not inhibitthe incision by ABCexcision nuclease of the fourth
step in vivo.In addition, we have noted a marked variation in phosphodiester bond 3' to a pyrimidine dimer (A. Sancar et
the rateof repair of specific pyrimidine dimers which appears al., 1984a). The fact that only about 10% of the total free
to be dependent upon both dimer type and the surrounding energy of photolyase-DNA binding is electrostatic at moder-
sequences.' ate salt concentrations has important implications for the
A strategy similar to that for determining k, is frequently specificity of binding and for the mechanism by which the
used to quantitate kl, i.e. reaction conditions are chosen such enzyme "searches" for its target.
that the term k, [ E S ]in Equation 6 is negligible; thus quan- At optimum conditions of ionic strength and pH, all for-
titation of [ES] as a function of time after mixing the reac- merly characterized sequence-specific DNA binding proteins
tants leads to an estimate of k, using the integrated rate (e.g. lac repressor and EcoRI restriction endonuclease) have
equation for a simple bimolecular reaction. K A values 2 10" M" (see Berg and von Hippel, 1985; Terry
et al., 1983), consistent with the selectivity of these proteins;
in contrast we find K A = 6 X lo7 M" for E. coli photolyase,
yet in vivo data indicate that the enzyme is highly specific.
Alternatively, if conditions cannot be found such that &[ES] Harm (1970b)reported that in E. coli cells containing approx-
is negligible then kl can be determined from the integrated imately 20 pyrimidine dimers and 20 photolyase molecules,
form of Equation 6: about 10 enzyme-substrate complexes werepresent at equilib-
rium. If we assume that all of the remaining enzyme molecules
2kl[ES], + b - J q ln-=tb-d q bound nonspecifically and that the photolyase binding site
In "
(9)
2k,[ES]t + b + J-q b + J-q spans 4 nucleotides (Jorns et al., 1985), then the minimum
specificity of photolyase binding (defined by the ratio of the
where
specific to nonspecific association constants) is 8 X 106/4 X
-b = kJS10 + kl[EIO + 4 (10) 10 = 2 X lo5 (assuming that each of the 8 X lo6 bases in the
and E. coli chromosome constitutes the beginning of a potential
nonspecific binding site). This value is similar to those re-
q = 4kz2[E]o[S]o- b2 (11) ported for sequence-specific binding proteins; thus it is the
Fig. 8 shows the kinetics of ES complex formation determined ratio of specific to nonspecific binding constants rather than
the absolute values of these constants that is important in
G . Myles, B. van Houten, and A. Sancar, unpublished observa- determining specificity (von Hippel and Berg, 1986). The
tions. major difference between E. coli photolyase and the lac re-
484 Binding of Photolyase to DNA
pressor or EcoRI is that the latter two proteins have large REFERENCES
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by electrostatic interactionswith DNA phosphates, and there- 14,131-160
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20,6929-6948
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von Hippel, 1977; Berg and von Hippel, 1985; Terry et al., tine, W. A. (1985) J. Biol. Chem. 2 6 0 , 11438-11441
1983; Jen-Jacobson et al., 1983). The S187 mutant of the Cohen, A., Fisher, W. D., Curtiss, R., 111, and Adler, H. J. (1968) Cold
EcoRI protein makes 2 phosphate contacts at pH 7.4 com- Spring Harbor Symp. Qwnt. Biol. 33,635-641
pared to 8 phosphate contacts for the wild type enzyme; the de Haseth, P., Lohman, L., and Record, M. T., Jr. (1977) Biochemistry
16,4783-4790
result is that while K A for the enzyme decreases from 10'' M-' Griffth, J. D., and Christiansen, G. (1978) Annu. Reu. Biophys.
(wild type) to 4.6 X los M" (S187), the enzyme retains its Bioeng. 7,19-37
specificity for binding to theEcoRI recognition sequence (Jen- Harm, H., and Rupert, C. S. (1968) Mutat. Res. 6,355-370
Jacobson et al., 1983), indicating that thespecificity of EcoRI Harm, H., and Rupert, C. S. (1970) Mutat. Res. 10, 291-306
binding, like photolyase, derives largely from nonionic inter- Harm, H. (1976) in Photochemistry and Photobiology of Nucleic Acids
(Wang, S., ed) Vol. 2, pp. 219-263, Academic Press, Orlando, FL
actions. Harm W., Harm, H., and Rupert C. S. (1968) Mutat. Res. 6,371-385
A number of sequence-specific DNA-binding proteins uti- Harm W. (1970a) Mutat. Res. 1 0 , 277-290
lize nonspecific electrostatic interactions to increase the ki- Harm W. (1970b) Mutat. Res. 10, 319-333
netic efficiency of binding; the high association rate constants Jen-Jacobson, L., Kurpiewski, M., Lesser, D., Grable, J., Boyer, H.
of proteins such as lac repressor generally exceeds the Smo- W., Rosenberg, J. M., and Greene, P. J. (1983) J.Biol. Chem. 2 5 8 ,
14638-14646
louchowski limit for diffusion controlled reactions (Riggs et Johnston, M., and Davis, R. W. (1984) Mol. Cell. Biol. 4, 1440-1448
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cating that theproteins find their targets by one-dimensional 2673-2679
or facilitated diffusion. Our results indicate that the contri- Jorns, M. S., Sancar, G. B., and Sancar, A. (1985) Biochemistry 24,
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Jorns, M. S., Baldwin, E. T., Sancar, G. B., and Sancar, A. (1987) J.
much less important for binding of photolyase than for other Biol. Chem. 261,486-491
DNA-binding proteins, as the bimolecular association con- Lohman, T. M., de Haseth, P. L., and Record M. T., Jr. (1980)
stant kl = 4.5 X lo6 "' s-' is well within the limit of $ Biochemistry 19,3522-3530
diffusion controlled reaction for a spherical protein of 50 A Madden, J. J., Werbin, H., and Denson, J. (1973) Photochem. Pho-
diameter. Furthermore, the minor contribution of electro- tobiol. 1 8 , 441-445
Patterson, M. C., and Rozzen, K. J . (1972) J. Bacteriol. 1 1 0 , 71-80
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use of unidimensional diffusion as a protein must have a Record, T. M., Jr., Lohman, T. M., and de Haseth, P. (1976) J . Mol.
significant level of nonspecific, electrostatic binding to slide Biol. 107,145-158
or track along DNA (Berg and von Hippel, 1985). The above Revzin, A., and von Hippel, P. H. (1977) Biochemistry 16,4769-4776
Riggs,A. D., Bourgeois, S., Newby, R. F., and Cohn, M. (1968) J.
arguments do not rule out relatively small contributions of Mol. Biol. 34, 365-368
nonspecific interactions to both the kinetics of photolyase Riggs, A. D., Bourgeois, S., and Cohn, M. (1970) J. Mol. Biol. 5 3 ,
binding and the intracellular equilibrium; indeed it is clear 401-417
that some nonspecific binding of the enzyme to DNA does Rupert, C. S. (1962) J.Gen. Physiol. 45,725-741
occur as minicells of E. coli which lack DNA are devoid of Rupert, C. S., Goodgal, S. H., and Herriott, R. M. (1958) J. Gen.
Physiol. 41,451-471
photolyase (Setlow and Cohen cited in Cohen et al., 1968) Sancar, A., and Rupert, C. S. (1978a) Nature 272,471-472
while minicells containing R factor DNA have a small but Sancar, A., and Rupert, C. S. (1978b) Gene (Amst.) 4, 295-308
detectable amount of photoreactivating enzyme (Paterson and Sancar, A., and Sancar, G. B. (1984) J. Mol. Biol. 172,223-227
Roozen, 1972). However, the magnitude of these effects rela- Sancar, A., Franklin, K. A., and Sancar, G. B. (1984a) Proc. Natl.
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tive to lac repressor or EcoRI must be small. Sancar, A., Smith F. W., and Sancar, G. B. (1984b) J. Biol.Chem.
It is of interest to compare the values which we obtain in 259,6028-6032
vitro for the kinetic and equilibrium constants for photolyase Sancar, G. B., Smith F. W.,Lorence, M. C., Rupert, C. A., and Sancar,
binding with values obtained i n vivo by Harm (1970a). Be- A. (1984) J. Bios!. Chem. 259,6033-6038
cause the photolysis reaction can easily be separated from the Sancar, G. B., Smith, F. W., and Sancar, A. (1985) Biochemistry 24,
1849-1855
binding reaction in vivo as well as i n vitro this enzyme offers Sancar, G. B., Jorns, M. S., Payne, G., Fluke, D. J., Rupert, C. S.,
a unique opportunity to measure reaction parameters under and Sancar, A. (1987) J. Biol. Chem. 261,492-498
both conditions and to compare them. Using the flash pho- Seawell, P. C., Simon, T. J., and Ganesan, A. K. (1980) Biochemistry
tolysis technique i n viuo, Harm (1970a) found kl = 1.1 X lo6 19,1685-1691
"1 s-l
, kz = 1.3 X to 1.9 X s-' from which K A can Segel, I. H. (1975) Enzyme Kinetics, pp. 109-111, John Wiley and
Sons, New York.
be calculated to be 8.5 X lo7 to 5.6 X 10' M-'. All of these Terry, B. J., Jack, W. E., Rubin, R. A., and Modrich, P. (1983) J .
values agree reasonably with the i n vitro values reported here: Biol. Chem. 258,9820-9825
kl = 1.4-4.2 X lo6 M-' s-', k2 = 2-3 X lo-' s-', and K A = 6.0 Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad.Sci.
X IO7 M - ~ .Thus we conclude that thekinetic and equilibrium U. S. A. 76,615-619
von Hippel, P. H., and Berg, 0.G. (1986) Proc. Natl. Acad.Sci.
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in vivo state. Winter, R. B., and von Hippel, P. H. (1981) Biochemistry 20, 6948-
finfin
""

Acknowledgments-We thank Drs. Peter von Hippel and OttoBerg Woodbury, C. P., Jr., and von Hippel, P. H. (1983) Biochemistry 22,
for their comments on the manuscript. 4730-4737
 Binding of Photolyase to DNA 485

By defmlfioo,
K P
pt i Pb+P i N l * ) +P. (eq. A16)
I+KAP

o r , since 1- 5 equals the filter bindlog rffxrcncy E