In this kinetic study,we further detail the reaction mechan-ism,showing that the overall ligation reaction is a super-imposition of two parallel processes: a processive ligation,in which
Trang 1Kinetics and thermodynamics of nick sealing by T4 DNA ligase
Alexey V Cherepanov* and Simon de Vries
Kluyver Department of Biotechnology, Delft University of Technology, the Netherlands
T4 DNA ligase is an Mg2+-dependent and ATP-dependent
enzyme that seals DNA nicks in three steps: it covalently
binds AMP,transadenylates the nick phosphate,and
cata-lyses formation of the phosphodiester bond releasing AMP
In this kinetic study,we further detail the reaction
mechan-ism,showing that the overall ligation reaction is a
super-imposition of two parallel processes: a processive ligation,in
which the enzyme transadenylates and seals the nick without
dissociating from dsDNA,and a nonprocessive ligation,in
which the enzyme takes part in the abortive adenylation
cycle (covalent binding of AMP,transadenylation of the
nick,and dissociation) At low concentrations of ATP
(< 10 lM) and when the DNA nick is sealed with
mismatching base pairs (e.g five adjacent),this
super-imposition resolves into two kinetic phases,a burst
ligation ( 0.2 min)1) and a subsequent slow ligation ( 2 · 10)3min)1) The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+ The activation energies of self-adenylation (16.2 kcalÆmol)1), transadenylation of the nick (0.9 kcalÆmol)1),and nick-sealing (16.3–18.8 kcalÆmol)1) were determined for several DNA substrates The low activation energy of transadeny-lation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction,and that the T4 DNA ligase–AMP complex is a high-energy intermediate To summarize current findings in the DNA ligation field,we delineate a kinetic mechanism of T4 DNA ligase catalysis Keywords: DNA ligase; end-joining; kinetics; mechanism of action; mismatching nick
T4 DNA ligase is an enzyme that catalyses formation of the
phosphodiester bond between the adjacent 5¢-PO4 and
3¢-OH groups of two dsDNA fragments [1] It is able to join
two dsDNAs (blunt-end or sticky-end ligation),or seal a
break between two ssDNA fragments annealed on the
complementary DNA strand (nick-ligation) It can join
phosphodiester linkages on triple-stranded nucleic acids [2],
seal single-stranded 1–5-nucleotide gaps [3],and act as a
lyase,removing apurininc/apyrimidinic (AP) sites in DNA
[4] The enzyme requires a bivalent metal cation such as
Mg2+or Mn2+,and joins DNA using ATP as a coenzyme
One phosphodiester bond in DNA is formed per ATP
molecule hydrolysed to AMP and pyrophosphate
The mechanism of catalysis of T4 DNA ligase comprises
three steps and involves two covalent reaction
intermedi-ates:
where ndsDNA is nicked dsDNA,AMP–ndsDNA is ndsDNA adenylated at the 5¢-phosphate of the nick,and
a one-sided arrow indicates that the reverse reaction is
at least three orders of magnitude slower than the
forward reaction
On the basis of the electrophoretic mobility shift assay experiments,it has been suggested that adenylated ligase forms transient Tcomplexes,E–AMPndsDNA,in search
of a phosphorylated 5¢-end of (n)dsDNA When the nick phosphate is found,it is adenylated,and a stable Scomplex
is formed,EAMP–ndsDNA [5] The enzyme in this complex has been suggested to stall on DNA until the nick is sealed and dsDNA is released Formation of the first phosphodiester bond during joining of the blunt ends has been proposed to happen accordingly; the main difference is the 2 : 1 dsDNA to enzyme stoichiometry in step 3 It has been suggested [5] that during blunt end joining,the ternary complex ligaseDNA is formed via two second-order associative reactions:
Scomplex þ dsDNA ! EAMPdDNAdsDNA Despite a good understanding of the overall reaction mechanism,relatively few articles have been dedicated
to the kinetic studies of catalysis performed by this enzyme The optimal concentration of Mg2+in the nick-joining reaction (8–10 mM),apparent K for the nicked
Correspondence to S de Vries,Kluyver Department of
Biotechno-logy,Delft University of Technology,Julianalaan 67,
2628 BC Delft,the Netherlands.
Fax: + 31 15 2782355,Tel.: + 31 15 2785139,
E-mail: s.devries@tnw.tudelft.nl
Abbreviations: ndsDNA,nicked dsDNA.
Enzymes: DNA ligase (EC 6.5.1.1).
*Present address: Metalloprotein & Protein Engineering Group,
Leiden Institute of Chemistry,Gorlaeus Laboratories,Leiden
University,Einsteinweg 55,PO Box 9502,2300 RA Leiden,
the Netherlands.
(Received 28 May 2003,revised 8 September 2003,
accepted 9 September 2003)
Trang 2dsDNA (1.5 nM),and ATP (14–20 lM) as well as the
apparent inhibition constant Ki for dATP (10–35 lM)
had been determined during the initial characterization
of the enzyme [6,7] It was shown that T4 DNA ligase
binds ATP covalently forming a lysine (e-amino)-linked
adenosine monophosphoramidate [8] Harvey et al [9]
have demonstrated that sealing of the pre-adenylated
nick in dsDNA is inhibited when T4 DNA ligase is
preincubated with ATP and Mg2+ Later it was shown
that the enzyme obeys Ping-Pong kinetics,and joins
dsDNA with multiple nicks in the nonprocessive mode
The true Kmfor ATP (100 lM) was determined in the
joining reaction with polydAd(pT)10as substrate (true
Km¼ 0.6 lM) [10] It was shown that T4 DNA ligase
seals nicks containing base pair mismatches [11–15],and
the effect of the ionic strength on the apparent Kmof the
mismatching dsDNA nick has been evaluated (e.g
200 nM at 0.2M NaCl vs 50 nM without salt)
[12,16,17] The pH-dependent equilibrium constant for
step 1 (0.0213 at pH¼ 7.0 and 25 C,pKa¼ 8.4) and
the standard free energy for cleavage of ATP to AMP
()10.9 kcalÆmol)1) have been determined [18] It was
shown that T4 DNA ligase is capable of synthesizing the
dinucleoside polyphosphates,such as Ap3A,Ap4A,
Ap4G,and Ap4dA,using ADP (d)ATP,GTP,and P3
as substrates [19,20] This secondary enzyme activity
may stem from the fact that ligase has two closely
located nucleotide-binding sites [21] Recently,a
dynamic mechanism of nick recognition by DNA ligase
has been proposed [22] The key feature of the
mechanism is the B-to-A DNA helix transition of the
enzyme-bound dsDNA motif,which results in DNA
contraction,bending,and unwinding For non-nicked
dsDNA,this transition is reversible,leading to
dissoci-ation of the enzyme For ndsDNA,this transition was
proposed to (a) trigger an opened–closed
conforma-tional change in the enzyme,and (b) force the motif to
accommodate the strained A/B-form hybrid
conforma-tion,the transition state in the nick-sealing reaction
In our previous work,we assessed the ability of T4 DNA
ligase to seal ndsDNA containing one to five adjacent
mismatching base pairs [14,23], aiming to use this enzyme in
the novel protocol of saturated scanning mutagenesis In all
cases,kinetic traces displayed pronounced biphasic
beha-vior,which was most spectacular with the nick containing
five base pair mismatches Apparently,this effect has not
been previously reported To understand its origin,we
decided to study the mechanism of T4 DNA ligase in more
detail We have previously reported a pre-steady-state
kinetic analysis of the first step of DNA ligase catalysis
(covalent binding of AMP [24]),showing that the enzyme
employs a two-metal-ion mechanism for this nucleotidyl
transfer reaction,using the dimagnesium ATP form
(ATPMg2) as a true substrate The monocoordinated
form,ATPMg,and/or free ATP bind DNA ligase
noncovalently,with Kd< 150 nM[21] Nucleotidyl transfer
is reversible,and the monomagnesium pyrophosphate form
MgP2O7 participates in the T4 DNA ligase-promoted
synthesis of ATP [24]
This work concentrates on the steady-state kinetic
analysis of the overall ligation reaction and an initial
thermodynamic characterization of the catalysis We aimed
to achieve the following goals: (a) to deepen the general understanding of the kinetic mechanism of the end-joining reaction,the biphasic behavior in particular,using an ndsDNA substrate with 5¢-mismatching base pairs; (b) to extract kinetic and thermodynamic parameters of the elementary steps of ligase catalysis; and (c) to summarize current findings on the mechanism of action of DNA ligase
in a single reaction scheme
Experimental procedures
Enzymes and oligonucleotides Three commercial batches of T4 DNA ligase were used,and were purchased from Amersham Biosciences (Uppsala, Sweden),Roche Molecular Biochemicals (Basel,Switzer-land),and MBI Fermentas (Vilnius,Lithuania) All enzyme batches showed similar activity and substrate specificity The proteins were essentially pure as judged by SDS/PAGE Protein concentration in the purchased enzyme stocks was determined using the BCA protein determination kit (Pierce Biotechnology,Rockford,IL,USA) Synthetic oligonucleo-tides were purchased with Eurogentec (Seraing,Belgium), except for the one labeled with the Cy5 fluorescent marker, which was obtained from Amersham Biosciences
Model system For studies on T4 DNA ligase-promoted repair of nicks in dsDNA,we used 72/24/(6–24)-mer synthetic DNA sub-strates Nonphosphorylated 72-mer B had the sequence 5¢-GTCCAAACAGCTATCTGCATCCGTCGACCTGC TCGGTTCCTTGGCTACACTGGCCGTCGTTTTACA ACGTCG-3¢ The 24-mer 5¢-DNA oligonucleotide C (5¢- here refers to the fact that the 5¢-oligomer is located upstream of the nick) had the sequence 5¢-CGACGTT GTAAAACGACGGCCAGT-3¢,and contained on the 5¢-end the fluorescent label Cy5 (Dye 667,No 27-1801-02; Amersham Biosciences),allowing easy quantification of the products of the joining reaction 3¢-DNA oligonucleotides (located downstream of the joining site) of different lengths (6–24-mers; Figs 1,2,3,4 and 8) were 5¢-phosphorylated; oligonucleotides M5C19,M1C6,and M4C7,in addition, contained base pair mismatches next to the joining site
Ligation of ndsDNA The ligation reaction was performed in 30 lL 66 mMTris/ HCl/1 mM dithiothreitol/0.05 mgÆmL)1 BSA,pH¼ 7.6 The buffer pH (measured at +20C) was adjusted to 7.19 (7.34) when the ligation was performed at +4C (+10 C), counting dpH/C ¼)0.026 for the Tris/HCl buffer pair To follow the formation of the adenylated DNA intermediate, [32P]ATP[aP] was used Concentrations of ATP,MgCl2, oligonucleotides,and the incubation temperature were varied according to the comments in the text ndsDNA was prepared by mixing the necessary amount of 72-mer oligonucleotide B,a stoichiometric amount of 5¢-Cy5-labeled 24-mer C,and the required donor oligonucleotide, followed by 5 min incubation at 65C,5 min incubation at
37C,and 10 min incubation at room temperature T4 DNA ligation buffer was added after pre-annealing of the
Trang 3oligonucleotide and before addition of the enzyme; the
reaction mixture was preincubated at the assay temperature
for 5 min The reaction was initiated by addition of the
enzyme Over time,aliquots of 0.5 lL were withdrawn and
mixed with 10 lL 100% formamide/10 mMNaOH/10 mM
EDTA/5 mgÆmL)1Blue Dextran,pH¼ 9.5
Stability of DNA ligase at ambient temperature
It is known that preparations of T4 DNA (RNA) ligase
gradually lose their activity when incubated at temperatures
above 0C [6,25] In this work, ligase was assayed at +4 C
for periods up to 25–40 h To avoid irreversible inactivation
of T4 DNA ligase during prolonged incubations,BSA was
added to the reaction mixture to a concentration of
0.05 mgÆmL)1,as reported previously [21]
Separation of ligation products and data analysis
All DNA separations were performed on an ALF Express
DNA sequencer (Amersham Biosciences) using 6–15%
acrylamide gels (Tris/borate/EDTA/7M urea) Usually,
runs were performed at 55C,with 80 mA current and
lasted for 1–3 h For each data point,the fluorescence of
two chromatographically separated peaks of starting
mater-ial and ligation product was obtained Separation of
[32P]DNA was visualized using a Molecular Dynamics
Phosphorimager SI (Amersham Biosciences) and quantified
using IMAGEQUANT software Data obtained from both
Cy5-labeled and32P-labeled DNA was imported intoIGOR
PRO version 4.0 (WaveMetrics Inc.,Salt Lake City,UT,
USA); further data analysis such as integration of peaks and
fitting was performed using the built-in functions of this
software package Rate values for the burst ligation were
determined as Vinit¼ F ¢[t]t¼ 0,where F [t] is the exponential
fitting function Steady-state rates were determined by
interpolating the steady-state region of the product
forma-tion curve with a linear regression funcforma-tion Numbers of
turnovers were calculated by dividing the initial
tion of ndsDNA in the reaction mixture by the
concentra-tion of T4 DNA ligase
Pre-steady-state kinetic analysis
Transient-state kinetic experiments were performed on the
Bio Sequential Stopped-Flow Reaction Analyzer SX-18MV
(Applied Photophysics,Leatherhead,Surrey,UK) using the
ozone-free 150 W xenon-arc light source The SX-18MV
software package for a single-wavelength operation
mode was used for the optical measurements Tryptophan
emission was excited at 280 nm and measured as the light
passing through a < 320 nm cut-off filter Kinetic traces of
protein fluorescence emission were obtained by averaging
three to ten shots Error estimates for the data values in
graphs and tables represent 95% confidence intervals
calculated using Student’s distribution function
In the stopped-flow instrument,we studied the
transient-state kinetics of the binding of Mg2+to the EATP complex
at different pH values For this experiment,a ligase solution
(7.5 lM,0.41 mgÆmL)1) was prepared by dilution of
a 10 mgÆmL)1 enzyme stock into 0.075 mgÆmL)1 BSA
solution in deionized water containing 1.5 m
dithioerythr-itol and 230 lM ATP (ATP was added from a 50-mM stock solution pre-equilibrated to pH¼ 7) This weakly buffered solution at pH 7.5 was stored on ice until further use A 150 mMTris/HCl buffer of the desired pH was prepared separately,as well as the 15 mMsolution of MgCl2in deionized water At 5 min before the mixing shots,
150 mM Tris/HCl buffer was diluted threefold into both enzyme and Mg2+ stocks Then 200-lL aliquots of the resulting solutions were withdrawn,mixed with each other, and the pH of the mixture measured In parallel,the solutions were pre-equilibrated to ambient temperature in the drive syringes and rapidly mixed in the stopped flow instrument,triggering the reaction
Results and discussion
Time course of the joining reaction
To study the T4 DNA ligase-promoted end-joining reaction,we used synthetic DNA oligonucleotides as described in Experimental procedures Two ndsDNA substrates were assembled: a substrate containing a 5-bp mismatching fragment at the nick (BM5C19),and a complementary nick substrate,BC24 T4 DNA ligase effectively utilizes both of these substrates (Figs 1,3 and 4)
Fig 1 Biphasic kinetics of joining of 3¢-oligonucleotides (M5C19, C24, M1C6, and M4C7) to 72/24-mer BC [dsDNA] was 1 l M ; [ATP] was 5.6 l M for M5C19,C24,and 1 m M for M1C6 and M4C7 Ligation of M5C19 and C24 was performed at +10 C as described in Experi-mental procedures M1C6 and M4C7 were joined at +4 C as pre-viously described [14] For joining of C24,the concentration of T4 DNA ligase in the assay mixture was 8 n M ,0.1 l M for M5C19,and 0.4 l M for M1C6 and M4C7 [dsDNA] (1 l M ) corresponds to the 125 turnovers of T4 DNA ligase in the case of C24 joining,10 turnovers in the case of M5C19,and 2.5 turnovers for M1C6 and M4C7.
Trang 4The mismatching nick (BM5C19) is sealed in two kinetic
phases (Fig 1A) In the case of C24,these phases are less
pronounced,and are observed only at low concentrations
of ATP (Fig 1B) Similar biphasic behavior is observed
with nicks containing one to four mismatching base pairs
(e.g Fig 1C,D) The origin of the two phases becomes
clear when the formation trace of the adenylated ndsDNA
intermediate is plotted together with the trace of the
ligation product (Fig 2) Virtually all ndsDNA is
conver-ted into AMP-dsDNA before 20% of the ligation product
is formed,and during this period the initial burst phase of
ligation takes place The slow ligation phase starts when
all available DNA substrate and T4 DNA ligase are
converted into their respective AMP-bound intermediates
The following mechanistic interpretation of the biphasic
kinetics is suggested During the burst phase,the enzyme
performs ligation processively,i.e by transadenylating
and sealing the nick without dissociating from the DNA
complex or being re-adenylated This process is not ideal:
a fraction of ligase molecules dissociates after the transfer
of AMP to the nick phosphate,rebinds AMP,and
performs another transadenylation step,which in parallel
to the processive ligation leads to the accumulation of
AMP–ndsDNA The slow steady-state ligation phase
starts when the concentration of the AMP–ndsDNA
intermediate reaches its maximum The overall end-joining
rate decreases because the adenylated DNA is ligated
either by the adenylated enzyme with a notably lower
rate,or,and what is more likely,by a small fraction of
the AMP-free enzyme (in agreement with previous data
[9,14]) It is also clear that the formation of the
phosphodiester bond rate-limits sealing of the
mismatch-ing nicks,and not the adenylation of the 5¢-nick phosphate For example,the rate of burst ligation of the oligonucleotide M1C6 (19 h)1) is,within experimental error,identical with the sealing rate of pre-adenylated AMP–M1C6 (18 h)1) [14]; adenylation of the oligonucle-otide M5C19 (1 min)1) is almost fivefold faster than the burst ligation (0.2 min)1)
A complex of T4 DNA ligase with AMP–ndsDNA is more stable in the case of a complementary nick ([5]),and the enzyme hardly dissociates from ndsDNA between the steps of transadenylation and nick-sealing As a result,the steady-state concentration of AMP–ndsDNA during liga-tion would be lower,and a difference in joining rates between the burst ligation and slow ligation phases at the same [ATP] is less pronounced For example,in the case of BC24,the rate of the burst phase is only about sixfold higher than the rate of the slow phase,in contrast with
> 100-fold difference in the case of the mismatching nick (Fig 1) The amplitude of the burst phase reaches 50% of the total extent of ligation when [ATP] is taken of the same
Fig 2 Formation of the ligation product (d) and the AMP–DNA
intermediate (s) during the joining of M5C19–72/24-mer BÆC Ligation
was performed at +10 C T4 DNA ligase (0.4 l M ) sealed ndsDNA
(1 l M ) in the presence of 1 m M ATP and 5 m M MgCl 2 Dotted traces
were obtained by fitting exponential functions to the experimental data
points [TP] 0 represents the number of turnovers required for the
joining of all ndsDNA in the reaction mixture.
Fig 3 Joining of C24to 72/24-mer BÆA at different concentrations of ATP (A) Product formation curves were computed by fitting single/ double exponential functions to the experimental data points [ATP] 0 for each curve is shown in the inset The linear/steady-state phase of the joining reaction is magnified in the inset Rate values were determined by fitting linear regression to the kinetic traces (fitted traces are shown in the inset) (B) (d) Turnover for the ligation of C24 and (s) M5C19 (burst ligation) at different [ATP] The 0–50 l M
region of ATP concentrations is magnified in the inset (C) Line-weaver–Burk plot of the (d) data shown in (B) Ligation was per-formed under the conditions described in Fig 1 The concentration
of Mg2+was 5.1 m M
Trang 5order of magnitude as [ndsDNA] in the reaction mixture,
e.g [ATP] 5 lM(Figs 2 and 4)
In general,biphasic kinetics are observed because the
ligase in complex with AMP–ndsDNA does not
re-adeny-late itself Otherwise,the enzyme would already be saturated
with AMP at the beginning of the reaction,irrespective of
the presence of ndsDNA,and would promote only slow
ligation In this sense,AMP–ndsDNA seems to shield
ligase from ATP Biphasic kinetics support the previous
proposals that: (a) the adenylyl moiety in the ligaseAMP–
ndsDNA complex occupies the ATP-binding pocket of the
protein [5],preventing a second nucleotide molecule from
entering the active site; and/or (b) ligase-bound ndsDNA
covers the ATP-binding site,hindering the access of ATP
from solution [26,27]
Effect of ATP concentration
The rate of the end-joining reaction catalysed by T4 DNA
ligase depends on the concentration of ATP in the reaction
mixture For example,the sealing rate of the complementary
nick in BC24 increases more than 10-fold with increasing
[ATP] from 5 to 400 lM,yielding Kapp
m (ATP)¼ 1.1 ·
10)4M (Fig 3),similar to the previously reported value
obtained with poly(dA)oligo(dT)10 [10] There are two
possible explanations for the decrease in the end-joining rate
at low [ATP]: either the binding of the nucleotide to the
ligase becomes rate limiting,or the equilibrium shifts
towards the nonadenylated enzyme From the
pre-steady-state kinetic analysis of ATP binding to T4 DNA ligase in
the absence of ndsDNA [24],both of these possibilities seem
unlikely At 5 mMMg2+,the rate constant for binding of
ATP is kon 9 · 105
M )1Æs)1,giving a binding rate of 4.5 s)1 at 5 lM ATP,which is more than 100-fold faster
than the observed rate of ligation at this [ATP] ( 1 min)1)
Therefore,binding of ATP could by no means limit the
enzyme turnover,unless inhibition by DNA is considered
Furthermore,the apparent Kdfor ATP in the noncovalent
EATP complex is below 150 nM[21],implying that at 5 lM
ATP the concentration of free enzyme in the absence of
ndsDNA is negligible; the ligase is essentially ATP bound
and/or AMP bound
Pre-steady-state kinetic experiments in which binding of
(n)dsDNA to T4 DNA ligase was studied suggest that this
is a rapid process with kobs 108M )1Æs)1(A Cherepanov,
D Pyshny & V Chikaev,unpublished results) Thus,
the binding of DNA at low [ATP] would be notably faster
than binding of ATP (102s)1for dsDNA vs 4.5 s)1for
ATP at 1 lM ndsDNA and 5 lM ATP) The situation is
reversed at high ATP concentrations,when binding of ATP
is faster than binding of DNA (102s)1 for DNA vs
4.5· 103s)1for ATP at 1 lMndsDNA and 5 mMATP)
Modeling studies indicate that ndsDNA in complex with
ligase hinders the access of solution ATP to the
nucleotide-binding site [26,27] Therefore, it is likely that the decrease in
the nick-sealing rate at low [ATP] occurs because DNA
prevents binding of ATP to the ligase This agrees with the
fact that the joining rate of the mismatching oligonucleotide
M5C19 does not decrease with [ATP] in the range 5 lMto
3 mM (Fig 4) In contrast,an approximately twofold
increase in the joining rate is observed,compared with
more than 10-fold decrease in the latter in the case of C24
In the case of M5C19,formation of the phosphodiester bond (< 0.2 min)1) limits the rate of the enzyme turnover, being slower than binding of a DNA substrate (102s)1), ATP (> 4.5 s)1),and joining of the complementary nick under the same conditions (> 1 min)1,Table 1) The increase in the joining rate of M5C19 at low [ATP] may indicate a shift of equilibrium towards the catalytically competent nonadenylated form of the enzyme
At [ATP] above 3 mM,inhibition of joining is observed:
at 10 mMATP the nick-sealing rate is roughly 20-fold lower than at 1 mM ATP (Fig 3) T4 DNA ligase is known to synthesize dinucleoside polyphosphates,such as Ap4A [19]
In this reaction the enzyme binds a second ATP molecule in the DNA-binding site with Kd0.1–0.25 mM[21]; the Ap4A synthesis is inhibited by ndsDNA [20] In our case,at low [ndsDNA] and high [ATP],the opposite situation may arise,when ATP inhibits binding of ndsDNA and subsequent ligation by occupying the dsDNA-binding site, leading to the synthesis of Ap4A (similar to that reported for GTP [20])
Fig 4 Joining of M5C19 to 72/24-mer BÆC at different concentrations
of ATP (A) Product formation curves at T ¼ 10 C Concentration of ATP is shown in the graph (h) 130 l M ATP; (j) 362 l M ; (s) 1.16 m M ; (d) 3.15 m M The burst-ligation phase of joining is magni-fied in the inset Reaction turnovers of the burst ligation (C) and of the slow ligation (D) are weakly [ATP]-dependent,increasing at [ATP] < 100 l M 2-fold The amplitude of the burst-ligation phase (B) increases more than 10-fold from 0.3 turnovers at 3.15 m M ATP
to 4.5 turnovers at 5.5 l M ATP,and the increase starts at [ATP] < 100 l M Ligation was performed under conditions equival-ent to those described in Fig 1 The concequival-entration of Mg 2+ was 5 m M [dsDNA] (1 l M ) corresponds to 10 enzyme turnovers (T4 DNA ligase was 0.1 l M ).
Trang 6In the case of joining of the mismatching oligonucleotide
M5C19,the amplitude of the burst phase increases more
than 10-fold from 0.3 turnovers at 3 mM ATP to 4.5
turnovers at 5 lM ATP (Fig 4),while the joining rate
remains roughly the same The amplitude of the slow phase
(extent of ligation) is independent of the concentration of
ATP,approaching 90% of the total concentration of the
ndsDNA substrate in the reaction mixture
Effect of Mg2+concentration
Mg2+is essential for DNA joining catalysed by T4 DNA ligase It has previously been shown that the optimal concentration of Mg2+in the ligation mixture is 10 mM [6] As shown in Fig 5A,two kinetic phases of the end-joining of M5C19 have different requirements for [Mg2+] The rate of burst ligation increases with [Mg2+],and the optimum is above 5 mM Mg2+ In contrast,the rate of the slow phase of joining reaches its maximum at 1 mM
Mg2+,above which joining is inhibited It is interesting that the dependence of joining rates on [Mg2+] follows the changes in the equilibrium concentrations of the mono-magnesium and dimono-magnesium forms of ATP (Fig 5B) The maximal rate of the slow phase of the joining of the mismatching oligonucleotide M5C19 corresponds to the maximal concentration of ATPMg On the other hand,the rate of the burst phase of joining of M5C19, and the joining rate of the complementary oligonucleotide C24 resembles more the increase in the concentration of ATPMg2
Temperature-dependence and pH-dependence
of self-adenylation of T4 DNA ligase
We have previously shown that self-adenylation of T4 DNA ligase in the absence of ndsDNA proceeds according to Scheme 1 [24]
Fig 5 [Mg 2+ ]-dependence of the joining of M5C19 (A) or C24(inset in
A) to 72/24-mer BÆC and the equilibrium concentrations of different ATP
forms (B) (A) Turnovers of the burst ligation for M5C19 (s),of the
slow ligation for M5C19 (d); inset in (A) ligation turnover of C24 (B)
Equilibrium concentrations of ATP forms were calculated using the K d
values in Table 2.
Table 2 K d values used to calculate equilibrium concentrations of ATP forms Concentration of the nicked dsDNA,1 l M (0.12 m M DNA phosphorus); ATP,1 m M ; T4 DNA ligase,0.1 l M in the case of M5C19 (8 n M in case of C24).
H + + ATP 4–
Mg 2+ + HATP 3–
« MgHATP 1– 6.6 · 10)3
Mg2++ ATP4–« MgATP 2–
8.9 · 10)6
Mg2++ MgATP2–« Mg 2 ATP 1.7 · 10)2 0.6Mg + dsDNA-PO 4
« Mg 0.6 dsDNA-PO 4
5 · 10)6 [39,40]
Table 1 Kinetic and thermodynamic parameters of T4DNA ligase catalysis (at 20 C, 5 m M Mg2+and pH = 7.6, [ATP] = 1 m M and [nds-DNA] = 1 l M ) Thermodynamic parameters were calculated by fitting Eqn (6) to the experimental data points,taking the transmission coefficient
j ¼ 1.
E A (kcalÆmol)1)
DS (calÆdeg)1Æmol)1)
a Mean values b Value relates to the slow nonprocessive ligation.
Scheme 1 Kinetic scheme of self-adenylation
of T4DNA ligase.
Trang 7T4 DNA ligase binds ATPMg noncovalently,but not
ATPMg2 Subsequently,EATPMg binds a second
Mg2+ion,forming a catalytic intermediate,EATPMg2
ATPMg2is the true substrate in the adenylation reaction,
while the monomagnesium pyrophosphate form,MgP2O7
is the true substrate for the reverse reaction, the synthesis of
ATP [24]
In this work,as part of an initial thermodynamic
characterization of T4 DNA ligase,the activation energy
of the adenylation reaction (cleavage of ATP) was
deter-mined in the absence of dsDNA using the stopped-flow
instrument To minimize the contribution of the reverse
reaction to the observed reaction rate,we excluded
pyro-phosphate from the reaction,mixing the EATP complex
with Mg2+ In the absence of excess pyrophosphate,
kapp2 contributes negligibly to kobs2 ,and kobs2 kapp2 ,and
2EA(obs)2EA(app) (for the k abbreviations see Scheme 1;
for the values of k¢ see [24])
In our experiments we used Tris/HCl buffer,which is
known for its pronounced temperature/pH-dependence
()0.026 pH units per C) To determine the activation energy
of self-adenylation,the reaction temperature was varied
between 10 and 30C,and pH therefore drifted by
0.5 unit We avoided using different buffering systems
because the kinetic parameters depend on the choice
of buffer For example,the use of Tris/maleimide instead of
Tris decreases the adenylation rate 1.5-fold (not shown)
pH-dependence
It is known that the adenylation of T4 DNA ligase strongly
depends on pH [18],because of the protonation of the
catalytic Lys159 To take into account the
temperature-induced pH drift of Tris/HCl buffer,we determined kobs2 at a
fixed temperature (20C) and different pH (6–9.5) In this
set of experiments,we used Tris/HCl buffer in part out of its
useful pH range,and special care was taken to measure pH
directly in order to account for the weakly buffering
components such as ATP,ligase and Mg2+ (see
Experi-mental procedures)
The stopped-flow traces recorded at different pH and
fitted values of kobs2 are shown in Fig 6 Eqn (4) was fitted to
the experimental data:
kobs2 ðpHxÞ kapp2 ðpHxÞ ¼ kapp2 Ka
Kaþ 10pH ð4Þ (pHx) is the pH-dependent rate constant of the
forma-tion of the enzyme–AMP adduct, kapp2 is the
pH-inde-pendent rate constant,and Ka is the protonation
constant of the 6-ammonium group of the catalytic
lysine residue
Interestingly enough,the determined pKa¼ 9.8 ± 0.3
for the protonation of the Lys159 is more than 1.2 pH units
higher than obtained in the equilibrium binding studies [18]
and 1 pH unit lower than the pKafor the e-amino group of
lysine in solution [28] This discrepancy between the results
in [18] and our data could possibly be explained by the
difference in the experimental conditions: buffer system
(Tris/HCl in our case vs Ches,Taps and Hepes for [18]);
concentration of Mg2+ (5 mM vs 1 mM); reaction
tem-perature (20C vs 25 C)
In the next set of experiments,we determined the kapp2 values at different temperatures (10–30C) and pH ¼ 7.6
at 20C Corresponding kinetic traces are shown in Fig 7
To correct the kapp2 values obtained for the temperature-induced pH drift,we employed the results of pH-depend-ence studies shown in Fig 6 The relation used for this purpose was as follows:
Fig 6 pH-dependence of the self-adenylation of T4DNA ligase Left: kinetic traces obtained at different pH (values are shown next to each trace) Right: the corresponding values of the observed rate constant
kobs2 The solid trace was computed by weighted fitting using Eqn (4), yielding values for the apparent pK a for the catalytic lysine residue and pH-independent kapp2 shown in the graph The reaction was started by rapid mixing of Mg2+solution with EATP complex pre-equilibrated
at the desired pH,resulting in the following final concentrations
of components: 2.7 ± 0.1 l M ligase,67.35 ± 0.07 l M ATP,
5 ± 0.05 m M Mg2+and pH values of 6.9,7.05,7.32,7.48,7.76,7.99, 8.28,8.6,8.79 and 9.2.
Fig 7 Kinetics of the self-adenylation of T4DNA ligase Fluorescence emission traces were recorded at different temperatures (values are shown in the graph) The reaction was started by rapid mixing of
Mg 2+
solution with EATP complex pre-equilibrated in buffer A for
5 min at temperatures of 11.3,13,14.8,16.5,18.4,20.2,22.1,24,26, and 27.6 C,resulting in final concentrations of components: 2.6 ± 0.1 l M ligase,71.42 ± 0.06 l M ATP,5 ± 0.05 m M Mg 2+
Trang 8kapp2 ðTxÞ
kapp2 ð20 CÞ¼
Kaþ 107:6
Kaþ 107:6þ0:026ðT x 20Þ ð5Þ
kapp2 (Tx) is the apparent rate constant at certain
tem-perature Tx, kapp2 (20C) is the value at 20 C,7.6 is the
pH of buffer A at 20C,and 0.026 is the pH drift of
Tris/HCl buffer perC
The Arrhenius plot of kapp2 ,corrected for the
temperature-induced pH changes is shown in Fig 8 The following
equation was used to fit the experimental data points:
ln kapp2
¼ ln jekT
h
DS
# 2
2EA
The plot is essentially linear with the slope of 16.7 ±
0.3 kcalÆmol)1(Table 1) Combining this value with the
reaction constants determined in [24],the Mg2+
-depend-ent and pH-independ-depend-ent rate constant of adenylyl
transfer at 37C can be estimated as 103)104s)1 This
indicates that at physiological pH,temperature and
[Mg2+],T4 DNA ligase binds ATP under strongly
suboptimal conditions,resulting in two orders of
mag-nitude lower reaction rates
Partial rates and activation energies of T4 DNA ligase catalysis
Joining of nicked dsDNA by T4 DNA ligase involves three catalytic steps: formation of the enzyme–adenylate,forma-tion of the ndsDNA–adenylate,and sealing of the nick These processes were studied at several temperatures,to estimate the activation energies of the individual reaction steps under the assumption that binding of the substrate(s) and dissociation of the product(s) do not limit the rate of catalysis Formation of the E–AMP intermediate was studied using the stopped-flow technique Synthesis of AMP–ndsDNA and sealing of the nick were monitored using [32P]ATP and/or Cy5-labeled DNA All three proces-ses yield essentially linear Arrhenius plots in the temperature range +4 to +30C (Fig 8) Nonlinearity for the joining
of the mismatching oligonucleotide M5C19 at high tem-peratures may be the result of the melting of the duplex AMP–M5C19BC
Formation of the E–AMP intermediate is the fastest measured process at all temperatures studied with a rate constant more than 10-fold higher than sealing of the complementary ndsDNA Both adenylation of T4 DNA ligase and sealing of the complementary nick have reason-ably high activation energies,which are identical within the experimental error (Table 1) In terms of the transition-state theory,the marked differences between the observed rates
of these reactions (i.e adenylation of the ligase and nick sealing) arise because of differences in the activation entropies (Table 1) The apparent activation energy for transadenylation of ndsDNA is 15 kcalÆmol)1lower than for adenylation of the ligase and/or sealing of the nick (Table 1; determined for the mismatching nick M5C19BC) It implies that the transfer of AMP to the terminal DNA phosphate is a thermodynamically sponta-neous reaction,and that the T4 DNA ligase–AMP complex
is a high-energy intermediate,as suggested in [18]
Kinetics of T4 DNA ligase catalysis
In a simple description,the nick-joining activity of T4 DNA ligase is a three-step enzymatic reaction which involves ndsDNA,ATP and an inorganic cofactor Mg2+ The reaction proceeds according to a Ping-Pong mechanism via formation of two intermediate products: E–AMP and AMP–ndsDNA [1,10]
In this work we observed the following phenomena: (a) biphasic kinetics of the nick-sealing,especially pronounced
at low [ATP] (Figs 1,2 and 4); (b) increase in the amplitude
of the burst-ligation phase at low [ATP] (Fig 4); (c) different [Mg2+]-dependence of each kinetic phase (Fig 5); (d) decrease in the end-joining rate at high and/or low [ATP] (Fig 3) To take these observations into account,
we produced Scheme 2
Biphasic kinetics According to Scheme 2,the initial burst-ligation phase results from the processive burst-ligation (route
1-…-6–7) In parallel to the processive ligation,a fraction
of ligase molecules enters a nonproductive adenylation cycle (route 1-…-6–1),aborting the catalysis between the steps of transadenylation and nick-sealing Abortive adenylation leads to the build-up of the AMP–ndsDNA pool and to the
Fig 8 Arrhenius plot of the individual steps of T4DNA ligase catalysis:
self-adenylation of the enzyme, transadenylation of the nick, and the
end-joining Trace 1,self-adenylation of the ligase The values of the
observed rate constant k obs
2 were corrected for the temperature-induced
pH drift of the Tris/HCl buffer using Eqn (5) Traces 2,joining of C6 to
72mer/24mer BC C6 to (BC) ratios are 1 : 1; 3 : 1; 10 : 1; 30 : 1, and
100 : 1 Trace 3,joining of C24 to 72mer/24mer BC C24 to (BC)
ratio is 30 : 1 Trace 4,adenylation of M5C19 in complex with 72mer/
24mer BC M5C19 to (BC) ratio is 2 : 1 Trace 5,joining of M5C19
to 72mer/24mer BC M5C19 to (BC) ratio is 2 : 1 The dotted traces
were obtained by fitting Eqn (6) to the experimental data points In the
case of trace 3 (joining of C24),and trace 5 (joining of M5C19),several
data points obtained at high temperatures were omitted to account for
melting of the DNA duplex.
Trang 9slowing of the ligation rate,because the adenylated enzyme
(4) does not seal preadenylated DNA The slow ligation
phase starts when the concentration of AMP–ndsDNA
reaches its maximum (steady-state conditions,Fig 2,
20–100 min)
The relative ratio of the processive nick-sealing and the
abortive adenylation depends on the quality of the ndsDNA
substrate In the case of a complementary nick,ligase forms
high-affinity complexes with the adenylated ndsDNAs [5],
and the kinetic contribution of the adenylation cycle to the
overall ligation is minor EAMP–ndsDNA complexes are
less stable in the case of a 5¢-mismatching nick [12,14], and
the contribution of the adenylation cycle pathway is more
significant
For example,the complex of ligase with
AMP-M5C19BC is unstable,transadenylation of M5C19 is
rapid ( 1 min)1),and the burst ligation is slow (0.2 min)1)
(Table 1) As a result,the slow ligation phase starts only
when all available ndsDNA substrate is converted: it is
either adenylated (via 5–6–1),or joined (via 5–6–7),and,at
the same time,the dominant enzyme fraction (4) is AMP
bound (Fig 2,20–100 min)
The adenylation of ligase is reversible: there is always a
fraction of the free enzyme (1) in the reaction mixture
formed via the routes 4–3–2–1,or 4–11–1) According to
Scheme 2,the slow ligation of M5C19 is performed by this
enzyme fraction via the route 1–6–7
Increase in amplitude of the burst phase at low
[ATP] At high [ATP] (1–5 mM),free ligase (1) binds
ATP (1–2) faster than it binds DNA (1–6) [(1–5)· 103s)1
for ATP vs 102s)1 for 1 lM ndsDNA] The repetitive
abortive cycling (1–2-…-6–1) leads to accumulation of the
AMP–ndsDNA intermediate,and its removal via the
nonprocessive route 1–6–7 is kinetically insignificant
Nonprocessive ligation 1–6–7 becomes significant only
during the slow ligation phase,when all available ndsDNA
substrate is adenylated,and the complexes 2–5 are no longer
productive
At low [ATP] (< 40 lM),ligase binds ATP slower than
DNA (< 36 s)1for ATP vs 102s)1for 1 lM ndsDNA)
During burst ligation,the free ligase (1) is thus engaged in
both processive ligation (1–2-…-6–7) and the
nonproces-sive scavenging of the preadenylated nicks via the route
1–6–7 Nonprocessive nick sealing (1–6–7) slows down the accumulation of AMP–ndsDNA,and the start of the slow ligation is delayed Delay of the slow ligation results in an increase in the amplitude of the burst-ligation phase (for M5C19 more than 10-fold; Fig 4) Similar results were recently reported [15] when a large number of the dsDNA substrates with one or two mismatching base pairs on both sides of the nick opposite tandem canonical bases were tested for ligation by T4 DNA ligase There,the highest ligation efficiency was observed at [ATP] of 10–100 lM
[Mg2+]-dependence The fact that the two ligation phases have different [Mg2+] optima stems,in terms of Scheme 2, from the [Mg2+]-regulated redistribution between the two enzyme forms: the adenylated ligase (4),and the free enzyme (1) E–AMPMg (4) is engaged in the processive ligation (4-…-7) An increase in [Mg2+] stimulates self-adenylation
3 fi 4,and inhibits the reverse reaction 4 fi 3 [24], causing the increase in 4,and,accordingly,the increase in the burst-ligation rate On the other hand,slow
nonprocessive ligation is performed by the free ligase (1) (route 1–6–7) The reverse reaction 4 fi 3 is the most efficient at 1–3 mM[Mg2+] [24],causing the increase in 1
As a result,the rate of the slow phase reaches its maximum
at this [Mg2+] When mismatching nicks containing tandem canonical bases at the site of ligation are sealed,the same narrow optimum of [Mg2+] between 1 and 3 mMhas been reported [15]
Decrease in end-joining rate at high and/or low [ATP] According to Scheme 2,the inhibition of ligation at high [ATP] occurs because T4 DNA ligase binds the nucleotide
at the dsDNA-binding site [20] with Kdbetween 0.1 and 0.25 mM[21] (routes 2–9, 3–10,and 4–11) The decrease in the rate of ligation at low [ATP] occurs because ndsDNA forms a complex with the ligase (route 1–8) From the modeling studies [26,27] one may conclude that, if the ligase
in complex (8) binds ATP at all,it will do so at a reduced rate The structural considerations are that ndsDNA in complex with T4 DNA ligase prevents access of ATP to the nucleotide-binding pocket of the enzyme,preventing ATP from either leaving or binding to the active site This is reflected in Scheme 2: ligase in complex with (n)dsDNA does not bind ATP at all
Scheme 2 Nick-joining by T4DNA ligase.
The rate constants k 1 –k 3 correspond to the
three steps of covalent catalysis.
Trang 10Scheme 2 only quantitatively addresses the
AMP-dependent reversal or inhibition of ligation Another
important assumption is that the ssDNA fragments to be
joined do not dissociate from the opposite DNA strand or
the EdsDNA complex The latter is certainly not the case
when short (4–12-mer) oligonucleotides are joined near their
Tm We further assumed that ndsDNA is present in the
Mg2+-coordinated form,neglecting the exchange between
the dsDNA-bound Mg2+ and K+ or Na+ at elevated
concentrations of the latter ions (> 100 mM) High
con-centrations of K+and Na+ inhibit DNA ligases [10,12,
29–31],perhaps,among other reasons,because ndsDNA
changes to the K+-(Na+)-coordinated form
In summary,this kinetic scheme of T4 DNA ligase
catalysis includes a two-metal-ion mechanism of ligase
adenylation,binding of the second nucleotide molecule at
the DNA-binding site,and synthesis of dinucleoside
tetra-phosphates,and treats the reaction in terms of processive
burst ligation and nonprocessive nick sealing
Physiological relevance of joining of mismatching
nicks by T4 DNA ligase
In vitro pre-steady-state kinetic studies performed in this
work suggest that T4 DNA ligase could rapidly adenylate
mismatching nicks in the infected cells,either at the late
stages of degradation of the cellular DNA or during the
subsequent replication of the coliphage T4 DNA: the ligase
gene is transcribed at steady levels throughout the eclipse,
reaching its maximum 3 min after infection [32] The sealing
of these pre-adenylated nicks,however,would be slow
because of relatively high intracellular [ATP] (1–3 mM) [33]
Under these circumstances,T4 DNA ligase would act on
the mismatching nicks more like an mRNA capping
enzyme,a member of the same superfamily of
nucleotidyl-transferases [34,35] In the cell, capping of the 5¢-end of
mRNA ensures protection from degradation by specific
exonucleases [36,37] In the case of capping the mismatching
nick,however,similar reasoning does not seem logical One
should consider that,in the cell at relatively high ionic
strength,both adenylation and joining of the mismatching
nicks could be suppressed to a large extent,as in the case of
in vitro ligations at 0.2M NaCl [12,16,17] Without
supportive experimental data in vivo,we will refrain from
assigning any physiological meaning to the low substrate
specificity of T4 DNA ligase reported here and in earlier
contributions [14,15,23] Instead, we demonstrate that this
unconventional activity of T4 enzyme is an invaluable tool
for elucidating the general kinetic mechanism of DNA ligase
catalysis
Conclusion
T4 DNA ligase-promoted end joining of nicked DNA is a
superimposition of two processes During the burst phase,
the main enzyme fraction performs ligation processively,i.e
by transadenylating ndsDNA and sealing the nick without
dissociation from the complex In parallel,a fraction of the
ligase molecules dissociates after transadenylation,and
rebinds ATP The slow ligation starts when most of the
ligase is AMP bound and the concentration of adenylated
ndsDNA reaches its maximal steady-state value The end
joining of AMP–ndsDNA during the slow phase is per-formed by a small fraction of the nonadenylated enzyme in a
nonprocessive mode The decrease in the rate of nick sealing
at low [ATP] occurs because dsDNA prevents binding of ATP to the ligase On the other hand,at low [dsDNA] and high [ATP],ATP inhibits binding of ndsDNA and subse-quent ligation by occupying the DNA-binding site
Acknowledgements
We thank Professor W R Hagen for critically reading the manuscript,and Dr P P Cherepanov for assistance with the32P experiments This work was supported by the Association of Biotechnology Centers in the Netherlands (ABON) (Project I.2.8) and in part by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW) (grant 349-3565).
References
1 Lehman,I.R (1974) DNA ligase: structure,mechanism,function Science 186,790–797.
2 Raae,A.J & Kleppe,K (1978) T4 polynucleotide ligase catalyzed joining on triple-stranded nucleic acids Biochemistry 17,2939– 2942.
3 Nilsson,S.V & Magnusson,G (1982) Sealing of gaps in duplex DNA by T4 DNA ligase Nucleic Acids Res 10,1425–1437.
4 Bogenhagen,D.F & Pinz,K.G (1998) The action of DNA ligase
at abasic sites in DNA J Biol Chem 273,7888–7893.
5 Rossi,R.,Montecucco,A.,Ciarrocchi,G & Biamonti,G (1997) Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action Nucleic Acids Res 25, 2106–2113.
6 Weiss,B.,Jacquemin-Sablon,A.,Live,T.R.,Fareed,G.C & Richardson,C.C (1968) Enzymatic breakage and joining of deoxyribonucleic acid VI Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacterio-phage T4 J Biol Chem 243,4543–4555.
7 Weiss,B.,Thompson,A & Richardson,C.C (1968) Ezymatic breakage and joining of deoxyribonucleic acid VII Properties of the enzyme-adenylate intermediate in the polynucleotide ligase reaction J Biol Chem 243,4556–4563.
8 Gumport,R.I & Lehman,I.R (1971) Structure of the DNA ligase-adenylate intermediate: lysine (e-amino)-linked adeno-sine monophosphoramidate Proc Natl Acad Sci USA 68, 2559–2563.
9 Harvey,C.L.,Gabriel,T.F.,Wilt,E.M & Richardson,C.C (1971) Enzymatic breakage and joining of deoxyribonucleic acid IX Synthesis and properties of the deoxyribonucleic acid adenylate in the phage T4 ligase reaction J Biol Chem 246, 4523–4530.
10 Raae,A.J.,Kleppe,R.K & Kleppe,K (1975) Kinetics and effect
of salts and polyamines on T4 polynucleotide ligase Eur J Bio-chem 60,437–443.
11 Harada,K & Orgel,L.E (1993) Unexpected substrate specificity
of T4 DNA ligase revealed by in vitro selection Nucleic Acids Res 21,2287–2291.
12 Wu,D.Y & Wallace,R.B (1989) Specificity of the nick-closing activity of bacteriophage T4 DNA ligase Gene 76,245–254.
13 Pritchard,C.E & Southern,E.M (1997) Effects of base mis-matches on joining of short oligodeoxynucleotides by DNA ligases Nucleic Acids Res 25,3403–3407.
14 Cherepanov,A.,Yildirim,E & de Vries,S (2001) Joining of short DNA oligonucleotides with base pair mismatches by T4 DNA Ligase J Biochem (Tokyo) 129,61–68.