Nucleic Acids Research
Volume 17 Number 12 1989
Confonrnational and thennodynamic consequences of the introduction of a nick in duplexed DNA
fragments: an NMR study augmented by biochemical experiments
Jane M.L.Pieters, Ruud
Comelis Altona*
M.W.Manst,
Hans van den Elst, Gijs A.van der Marel, Jacques H.van Boom and
Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden and 'Department of
Biochemistry, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
Received March 17, 1989; Revised and Accepted May 18, 1989
ABSTRACT
NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers:
d(ACGGCT) (I), d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III),
d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable
duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY
experiments of MI and M2 revealed that these fragments form B-DNA type duplex structures. A
comparison of the chemical-shift data of the nicked duplexes with those of the intact duplex of III
+ IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The
chemical-shift profiles of Ml and M3 were used to obtain the thermodynamic data for the duplex/coil
transitions. The profiles of MI were analysed by means of a new thermodynamic model (TRIDUP).
From the calculated thermodynamic data of MI and M3 it is concluded that the melting behaviour
of MI occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate
(M2) was almost completely joined after an overnight incubation at 14 'C.
INTRODUCTION [1,2]
The role of DNA ligases is well established in DNA replication, in DNA repair and in
genetic recombination in prokaryotic and in eukaryotic cells [3]. These enzymes catalyse
the restoration of an interruption of a single strand in double-helical DNA. For the joining
activity the enzyme requires juxtaposed 3'-hydroxyl and 5'-phosphoryl ends aligned in
a duplex structure [4]. The enzymes obtained from unaffected and from T4-infected E.
coli have been investigated most thoroughly [4]. However, until now little information
is available concerning structural details of the nicked duplex structure.
The present work describes an NMR study, augmented with biochemical experiments,
of a synthetic nicked duplex structure. The compound consists of a 12 base-paired duplex
that features an interruption in the centre of one strand of the double helix. The
conformational properties of the nicked duplex are compared with those of the intact doublehelical fragment. Furthermore, the influence of removal of the phosphate at the interruption
is demonstrated. Thermodynamic analysis of duplex formation of the nicked as well as
of the intact duplex structure is used to study the amount of cooperativity of the two hexamer
strands in the melting behaviour of the nicked duplex. Finally, the T4 polynucleotide ligase
activity upon this synthetic nicked duplex fragment is reported.
MATERIALS AND METHODS
The DNA compounds d(ACGGCT) (I), d(TGCAGT) (II) and d(AGCCGTACTGCA) (III)
were synthesized via an improved phosphotriester method as described elsewhere [5,6].
The 12-mer d(TGCAGTACGGCT) (IV) was synthesized via a solid phase approach [7],
t IRL Press
455 1
Nucleic Acids Research
1
2 3 4 5 6 7 8 9 10
12
G-C-A)
5d(A-G-C-C-G-T-A-C-Tx
* *
x * - x
3d(A-C-G- T- C-A-T-G-C-C -G -A)
II
irI
MM
1 2 3 4 5 6 7 8 9 10 11 12
5'd(A-G-C-C-G-T-A-C-T-G-C-A)
31d(T-C-G-G-C-A)
24 23 22 21 20 19
J
M4
J
M5
2 3 4 5 6 7 8 9 10 11 12
5'd(A-G-C-C-G-T-A-C-T-G-C-A)
3'd(T-G-A-C-G-T)
,
18 17 16 15 14 13
1 2 3 4 5 6 7 8 9 10 11 12
Vd(A-G-C-C-G-T-A-C-T-G-C-A)
3'd(T-C-G-G-C-A)(T-G-A-C-G-T)5'
24 23 22 21 20 19 18 17 16 15 14 13
I +
Scheme I. Numbering of the residues in the various duplex structures.
The numbering of the residues in compounds M2 and M3 corresponds with that in Ml: in M2 sequence I is
5'-phosphorylated at residue dA(19); in M3 Iand flare linked by a phosphodiester bond to form the intact duplex.
whereas the 5'-phosphorylated DNA hexamer d(pACGGCT) (Ia) was synthesized via a
modified phosphotriester procedure [8]. After purification the compounds were treated
with a Dowex cation-exchange resin (Na+-form) to yield the sodium salts.
NMR spectroscopy
NMR-samples were lyophilized three times from D20 (99.75 %) and finally taken up in
400 1l D20 (99.95 %). A trace of tetramethylammonium chloride (Me4NCl) was added
as an internal shift reference and EDTA (0.1 mM) was added to neutralize paramagnetic
impurities; pH values were adjusted to 6.5 -7.5 (meter-reading). The chemical shift of
Me4NCl relative to sodium 4,4-dimethyl-4-silapentanesulphonate (DSS) is 3.18 ppm at
25°C. The non-exchangeable protons of III (MM) (Scheme I) were studied at 7 mM singlestranded oligonucleotide concentration. Furthermore, several mixture samples of the DNA
compounds were prepared containing 7 mM of DNA for each strand: Ml: I + I + III;
M2: Ta + II + III; M3: III + IV; M4: I + III; MS: II + III (Scheme I). In order to
obtain equimolar mixtures of the DNA compounds, the concentrations of the separate single
strands were determined spectrophotometrically and the solutions were mixed in the correct
ratio. The NMR samples that were used in the nuclear Overhauser effect (NOE) experiments
were degassed and sealed under dry nitrogen.
In order to observe exchangeable-proton resonances of the mixture samples (Ml -M5)
and of MM a second set of NMR samples was prepared in 90/10 H20/D20 solvent
mixtures. The DNA concentrations were identical to those of the D20 samples and the
pH values were adjusted to = 6.5 (meter-reading). DSS was used as an internal reference
for the imino-proton signals.
IH-NMR spectra were recorded on a Bruker WM-300, a WM-500 and on an AM-500
4552
Nucleic Acids Research
p.024.8.4.604.4
4
2240
~~~~~~~~~~~2.2
S
9
*16
__
e21
_
_A
_
~
~
-
.
19~~~~__ _ _-- ~~~~~~~~2
- Q .6
19
~
114
.
3
23~
_
_
_
_
_
1
7
1
0
2.8
3.0
%~~~~~~~~~~~
2
~~~~~~~~~~~~~~~~~~~~3.
0
PPM
5.0
4.8
4.6
PPM
4.4
4.2
4.0
Figure 1. Part of the NOESY spectrum of M2; 21 mM single-stranded DNA, 295 K, pH 7.2, tm = 400 ms.
The region in which H8/H6-H1 '/H5 connectivities are found is shown. The broken line represents the interresidue
NOE between T(6) and dA(7).
spectrometer. The WM-300/500 instruments were equipped with an ASPECT-2000
computer, whereas the AM-500 spectrometer was interfaced with an ASPECT-3000
computer. In the case of the D20 samples the residual HDO peak was suppressed by
irradiation at the HDO frequency. The intensity of the H20 signal in the imino-proton
spectra was reduced by means of a time-shared long pulse in combination with a data-shift
accumulation routine [9,10].
NOESY experiments were performed essentially as described elsewhere [11-13].
NOESY experiments were recorded for Ml, M2 and M3 at 295 K. Mixing times were
varied between 400 and 600 ms. In all experiments 512 4K FIDs were collected. Before
phase-sensitive Fourier transformation, both time-domains were apodized with Gaussian
windows and the tl domain was zero-filled to 2K.
In order to study chemical shifts as a function of temperature series of one-dimensional
spectra were recorded at 300 MHz at several temperatures between 5 °C and 95 'C. Exact
sample temperatures were determined from the chemical shift of the residual HDO signal
[14]. The conformational notation and the numbering of the residues accord with the latest
IUPAC-IUB recommendations [15,16].
4553
Nucleic Acids Research
UV spectroscopy
The samples used for the ultraviolet-melting experiments of M2 were prepared by dilution
of the NMR sample to = 30 zM (total single-strand concentration) with H20; MgCl2 was
added up to the required concentration. Melting curves were recorded at 260 nm on a
Cary 118C spectrophotometer, interfaced with a MINC computer [17]. The samples were
heated at a rate of approximately 1 degree/minute.
Phosphorylation and ligation
The enzymes T4 polynucleotide kinase and T4 DNA ligase were purchased from
Pharmacia. [-y-32P]ATP (3000 Ci/mmol) was purchased from Amersham.
The 5' terminal of the synthetic DNA oligomer I was phosphorylated in a 20 td reaction
mixture containing 50 mM Tris-HCl (pH = 7.6), 10 mM MgCl2, 5 mM dithiothreitol,
0.1 mM spermidine, 0.1 mM EDTA, 20 IACi [Ly-32P]ATP, 15-100 ltM I and 3 units of
T4 polynucleotide kinase [18]. After incubation at 37 °C for 30 minutes, the reaction mixture
was extracted once with phenol/ chloroform/isoamylalcohol (25:24:1); 5 itg of tRNA was
added as precipitation carrier and the mixture was precipitated twice in 2 M
ammoniumacetate by means of ethanol. The phosphorylated oligomer (Ia*) was dissolved
in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA.
Ligations were performed in 80 I1 reaction mixtures containing 20 mM Tris-HCl (pH
= 7.5), 22.5 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 2-10 zIM DNA and 2 units
of T4 DNA ligase [18]. Incubation was carried out at 14 °C and 10 IL aliquots were taken
at times from 0-24 hours. The ligation products were analysed on 20 % polyacrylamide,
8 M urea slab gels.
RESULTS AND DISCUSSION
Assignment of the non-exchangeable proton resonances
The assignments of all base, Hi', H2' and H2' proton resonances, except the H2 signals
of Ml, M2 and M3, were based upon an analysis of NOESY spectra and followed established
procedures [19,20]. The numbering of the residues in the various duplexed structures is
represented in Scheme I. The structural information obtained from the analysis of the
NOESY spectra can be used to determine whether or not the duplex is distorted by the
presence of a single-stranded break.
The region of the NOESY spectrum of M2 (295 K, Tm = 400 ms) that contains the
connectivities between the base H8/H6 and HI' sugar signals is displayed in Figure 1.
This part of the spectrum as well as the base-proton/H2'-H2" connectivities were used
in the sequential assignment method. First the assignment based upon inter- and intraresidue
base(n)-H1'(n- 1) and base(n)-Hl'(n) NOEs of the dodecamer strand of the duplex was
completed. Note that no interruption of the NOE pattern was observed between the residues
T(6) and dA(7), which are located opposite the nick in the other strand (Figure 1).
Next, the sequential assignment of the two hexamer strands was undertaken. In the
assignment procedure of the hexamers it is convenient to start the assignment from the
H6 proton of the 5'-terminal residue T(13) of II. This proton could be located by the
observation of the intraresidue H6/CH3 connectivity in combination with the absence of
an interresidue CH3(n) -H6/H8(n- 1) NOE. The sequential assignment was continued up
to residue T(18), the 3' terminus of II. At this point an interesting NOE connectivity is
noted between H1'(18) and the H8 signal of dA(19), the 5'-terminal residue of Ia (Figure
1). Interresidue NOEs between H8(19) and H2'(18) and between H8(19) and H2"(18)
are also observed (not shown). This indicates that the duplex structure is conserved at
4554
Nucleic Acids Research
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4555
Nucleic Acids Research
the nick in a more or less regular fashion. The remaining base-proton and sugar-proton
resonances (residues dC(20)-T(24)) were assigned in a similar sequential manner
(Figure 1).
The same assignment procedure was employed to assign the base-proton and sugar Hi',
H2' and H2" signals of Ml and M3. Unfortunately, severe overlap in the NOESY spectrum
of Ml prevents an unambiguous decision concerning the question whether or not interresidue
NOEs appear at the nick between the residues T(18) and dA(19).
The observation of the relatively strong intraresidue base(n) - H2'(n) and interresidue
base(n) -H2 "(n - 1) NOEs of all mixtures indicates that Ml, M2 and M3 predominantly
adopt a right-handed B-type duplex structure at low temperature [21 ]. The chemical-shift
data of the assigned signals are listed in Table I.
Inspection of Table I reveals that the presence of a single-stranded break causes some
structural changes located predominantly in the base-pairs that flank the nick. The largest
shift changes are found when the intact duplex (M3) is compared to the nicked duplex
(Ml) in which the phosphate between T(18) and dA(19) is absent. Pronounced upfield
shifts (DM3 -6M1) are seen for H8 and the sugar-proton signals of dA(l 9) (H8: 0.52 ppm;
Hi': 0.11 ppm; H2': 0.28 ppm; H2": 0.21 ppm) and for H2"(18) and Hl'(20), 0.15 ppm
and 0.29 ppm, respectively, relative to the intact duplex structure (M3). At the same time
significant downfield shift changes (6M3-6M1) are displayed by the HI' and H2'
resonances of T(18), -0.32 ppm and -0. 16 ppm, and by H6(20) and H2'(20), -0.16
ppm and -0.18 ppm, respectively. For M2, having a doubly ionized phosphate in the
region of the nick, the shift differences 6M3 -6MI are much smaller, in general . 0.1 ppm,
except H ', H2' and H2 " of T( 18).
The present chemical-shift data suggest that a break in one of the two strands of B-type
double-helical DNA causes only small distortions of the duplex structure, whereas the Btype duplex structure is mainly retained. It appears likely that local conformational changes
at the break as well as in the opposite strand are reduced by the presence of a
5'-phosphorylated terminus at the interruption.
In contrast with the observations described above, the 12 + 6 mixtures M4 and MS
show a different behaviour. At low temperature the spectra of M4 as well as of MS display
two distinct sets of resonances (not shown) with differing intensities, i. e. a minor and a
major species occur side by side, presumably in (slow) conformational equilibrium. From
dilution experiments it could be concluded that both sets of signals correspond to dimeric
species. Pertinent clues as to the identity of the two species were obtained as follows:
investigations of the IH-NMR spectra of pure III at high DNA concentration (7 mM) and
low temperature revealed that the dodecamer adopts a mismatched duplex structure (MM),
consisting of eight Watson-Crick type base pairs, two C x T mismatches in the core and
Ax A mismatches at the termini (Scheme I). A comparison between the spectrum of MM
and those of M4 and MS demonstrated that the minor set of resonances in the spectra of
the two mixtures can be ascribed to the mismatched duplex structure MM. The set of signals
that predominates the spectra is identified with the 12 + 6 duplex structures of I + III
and II + III, respectively (Scheme I). The maximum amount of the mismatched species
is reached at 278 K: M4 85 %, MM 15 %; MS 70 %, MM 30 %.
Chemical-shift profiles and thermodynamic analysis
In order to study the thermodynamic behaviour of the nicked (Ml) as well as of the intact
dodecamer duplex structure (M3), chemical-shift profiles were constructed for both
mixtures. Moreover, a thermodynamic analysis of MI gives an insight into the amount
4556
Nucleic Acids Research
M(24)
<8(22)
4.5
6
_
6(24)
8(17)
M (6)[95M(13)
T~
-2.0
300
350
TEMPERATURE
300
350
TEMPERATURE(
Figur 2. Chemical-shift vs. temperatre pr-ofiles of some base-pr-oton resonances of M3. Plotted curves are calculated
according to the DUPSTAK procedure (see text). For the sake of clarity, one half of the measured points is omitted.
Of cooperativity of the two hexamer fragments in the formation of the nicked duplex
structure. This cooperativity of duplex formation is to be understood as a reversible transition
in which the two hexamner strands combine simultaneously with the dodecamer to form
the duplex MI (Scheme I).
s that ethe tspectra
o
In the previous section it was ope
mentioned
sM. Ploted cs are5 display
wof
(seerte re region, whereas in the spectrum of MI only
two sets of theDUw-Teme
signals in
one set of resonances is observed. This observation appears an imnportant clue to the presence
of cooperativity of duplex formation of the nicked duplex structure. This can be seen as
follows: when significant cooperativity of the two hexamer strands would be absent the
population of the nicked duplex structure Mlcan be calculated from the observed molar
fraction duplex species I +). III and + I11 in the spectra of M4 and M5, respectively.
In that mode a population nicked duplex is estimated to be about 60 % at 278 K, contrary
to experiment (100 % at 278 K). Moreover, in the absence of significant cooperativity
of the two hexamer strands one expects that the spectrum of MI at low temperature should
show two setsrnesobser,
of
but only a single set is observed.
7he intact dodecamer duplexf
(oM ). Chemical-shift profiles were constructed for all H8
and thymine H6 and CH3oresonances of M3 at 14 mM single-strand DNA concentration.
Most of the shift profiles show a sigmoidal shape that reflects the shift of the
duplex/monomer equilibrium with the temperature. The low-temperature plateau
corresponds to the intact duplex form, whereas the high-temperature plateau region
represents the monomer (Figure 2).
These profiles were analysed by means of a non-linear least-squares fitting procedure
4557
Nucleic Acids Research
M(24)
M(9)
MO 8)
-20
4.0-
6(6)^
6
24
(
320
TEMPERATURE (K)
270
370
320
TEMPERATURE (K)
370
Fgue 3. Chemical-shift vs. temperature profiles of some base-proton resonances of M1. Plotted curves are calculated
according to the TRIDUP model (see text). For the sake of clarity, one half of the measured points is omitted.
(Hartel et al., unpublished results, see also [ 14]) in combination with a recently developed
three-state model DUPSTAK [22]. In the DUPSTAK model the relation between the
observed chemical shift, bobs, and the temperature is given by:
(1)
6obs=6coil+Px Ax+Pd Ad
in which &,Oil represents the chemical shift of a given proton in the random-coil state; px
and Pd stand for the population stacked species and duplex, respectively; Ax and Ad
correspond to the chemical-shift increment in the stacked single-stranded form and in the
duplex relative to the random coil, respectively. The relation between the molar fractions
(px and Pd), the thermodynamic parameters (T.X, Td, AS' AS) and the total strand
concentration is given elsewhere [231.
The chemical-shift profiles of some base-proton resonances of M3 are shown in Figure
2. Most of the shift profiles show pronounced chemical-shift changes in the low-plateau
region. These shift changes are generally ascribed to small and local conformational
transitions in the intact duplex [23,24]. To account for these shift changes a linear
temperature dependence of the chemical shift in the duplex (AAd) was introduced:
Ad = Ad'+ (T-273) AAd
(2)
In equation (2) Ad' corresponds with the chemical-shift increment of a given proton in
going from the random-coil form to the duplex at a chosen temperature limit (273 K).
The analysis of the shift profiles with the aid of the DUPSTAK model rests in part upon
knowledge of the thermodynamic data for single-helical stacking. These data were not
available for the compounds at hand. Therefore, a mean value for the stacking parameters
-
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Nucleic Acids Research
C
Kd
1/3 D
4
Kx
No
X
Scheme H. Three-state equilibrium as used in the TRIDUP model; random coil (C), single-stranded stack (X)
and duplex (D).
is taken (T. 312 K, AS' -89 J-mol-'K-') based upon thermodynamic analysis of a
number of small single-stranded oligonucleotides [22,23,25,26]. This choice appears
acceptable for several reasons: (i) it is known that the stacking data of a given residue
in a single helix are determined mainly by the neighbouring bases [22,26]; (ii) the spread
in the thermodynamic parameters AS' and AHX appears small relative to the expected
values for duplex formation.
In the preliminary calculations all parameters, except T. and AS', were allowed to
relax. These calculations resulted in small deviations in ASdo (28 J * mol -K1) and TnA
(4- 1 K) values. Since these variations appear of no statistical significance, the entropy
term was constrained to adopt the mean value: ASO = -984 J mol-PK-'. In the final
analysis an average Td value of 361 K (standard conditions, 1 M single-stranded DNA)
and an average AHI value of -356 kJ mol-I were calculated for the duplex/coil
equilibrium of M3. As an example of the satisfactory accuracy of the fit, Figure 2 shows
the observed chemical shifts of some base-proton resonances together with the calculated
profiles by means of the DUPSTAK model.
The nicked duplex (Ml). Chemical-shift profiles of all H8 and thymine base-proton signals
of Ml were constructed at 21 mM single-stranded oligonucleotide concentration (7 mM
duplex). Most shift profiles show a sigmoidal behaviour, representing the duplex/coil
transition. Again, the high-temperature plateau is identified with the random-coil state,
whereas the low-temperature plateau corresponds with the intact duplex, Figure 3^.
For the melting transition of Ml two a priori different processes can be envisaged. In
the first model, the duplex/coil equilibrium occurs fully cooperatively and in the second
one, the two hexamers independently form a duplexed structure witi III. These two extreme
ways of melting behaviour of Ml should be considered in the analysis of the shift profiles.
In the previous section it was demonsra that the melting transition of Ml occurs probably
as a more or less cooperative process. In the first calculations the profiles were analysed
by means of a fully cooperative model.
In that case the well-established procedures [22,23,27,28] cannot be used to determine
thermodynamic data, because the nicked duplex consists of three strands instead of the
regular two (self-)complementary strands. Therefore, a new model TRIDUP was explored.
In the TRIDUP model three species: random coil (C), single-stranded stack (X) and duplex
(D) occur in equilibrium (Scheme II). It should be noted that in this model the stacking
proclivity of single-stranded fragments is taken into consideration. In an earlier paper [22]
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Nucleic Acids Research
we demonstrated that it is necessary to take into account single-stranded stacking to describe
the melting transition of the duplex to coil.
In the TRIDUP model the observed chemical-shift vs. temperature is given by equation
(3). In analogy with the DUPSTAK model a temperature-dependent duplex shift was
incorporated:
6obs=6coil +PxAX+Pd (Ad' + (T-273) A\Ad)
(3)
The symbols have been defined above. The equilibrium constants Kd and Kx follow from
the usual relations:
K XI
[D]
Kx [C]
(4)
[C]3
The relations between the equilibrium constants, the total strand concentration co and the
populations are given by:
I +Kx)
Kd(
Q 9Kd Q )
Pd = c co
(-g
Px
Kx
*
c
((CO)2
Q
(6+K
Q
l + Kx)
-9Kd
Q
I + Kx 3 c2 0
6Kd
9K ))
(5)
1
V3
Note that in the case of complementary fragments co is taken as one ninth of the
stoichometric concentration.
In the first analysis of the shift profiles with the aid of the TRIDUP model the stacking
parameters (Tmx: 312 K, AS': -89 J mol-'K-') were constrained. The remaining
parameters were minimized simultaneously. Rapid convergence was reached for all shift
profiles that display relatively large chemical-shift changes in the duplex-to-coil transition
(0.14 ppm). The calculations yielded a small dispersion in AS' ( 28 J mol- K-') and
Tmd (±2 K) values. Since the error analysis indicated that these variations were not
statistically significant in the final calculations, the entropy term was constrained to adopt
the weighted-mean value obtained for the various shift profiles: AS' = -869
J mol-'K-'. The final analysis reveals an average Tmd value of 359 K (standard
conditions) and a AH11 value of -312 kJ mol'- for the coil/duplex equilibrium. At the
experimental concentration a melting temperature of 45 °C is obtained. The satisfactory
fit between the experimental data and the calculated shifts by means of the TRIDUP model
is illustrated in Figure 3.
At this point it is of interest to compare the experimental thermodynamic data of Ml
with those obtained for M3. The parameters AS' and AI-5 mutually agree although the
nicked duplex is slightly destabilized relative to the intact duplex. The decrease in enthalpy
(44 LJmol-l) as well as in entropy (115 J-mol-'K-') roughly corresponds to a
destabilization expected for the loss of a single base pair [29]. Therefore, it is concluded
that the melting behaviour of the nicked duplex structure occurs as a cooperative transition.
4560
Nucleic Acids Research
A
B
E
14.0
PPM
12.0
Figure 4. Imino-proton resonances of the various mixture samples at 278 K, pH 6.5.
A MS, 14 mM single-stranded DNA concentration;
B M4, 14 mM single-stranded DNA concentration;
C Ml, 21 mM single-stranded DNA concentration;
D M2, 21 mM single-stranded DNA concentration;
E M3, 14 mM single-stranded DNA concentration;
Signals indicated by * correspond to the minor mismatched duplex form of III.
The same conclusion can be drawn by means of a different route, as follows: in the
absence of significant cooperativity of the two hexamer compounds in the duplex/coil
transition of Ml the duplex/coil equilibrium consists of two independent duplex/coil
equilibria viz. I + III and H + III. Thus, the obtained thermodynamic data should agree
with those expected for the formation of a duplexed structure comprising of six base pairs.
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In order to investigate this possibility the shift profiles of MI were analysed by means
of the DUPSTAK model. The least-squares fits of the shift profiles were carried out
analogously to those described for M3. The AS' value was constrained at -715
J molK-IK the weighted-mean value obtained for the various shift profiles. The analysis
resulted in an average Tmd of 342 K and AHI value of -244 kJ mol'. These values
differ substantally from the expected thermodynamic parameters, AHS -175 kJ mol-I
and ASd =-440 J mol -'K' (mean values for a duplex consisting of six base pairs,
taken from reference [29]).
Thus, the analysis by means of the DUPSTAK model reinforces our conclusion that
the melting transition of a duplex containing a singlestranded break occurs cooperatively.
The exchangeable proton resonances
The imnino-proton spectra of Ml to MS at 278 K are depicted in Figure 4. These spectra
clearly demonstrate that all duplex structures are fiully formed at low temperature. The
imino-proton resonance that corresponds to the A* T base pairs in the mismatched duplex
structure of IIl is marked by an asterisk. This inino-proton signal is assigned by comparison
with the inilnospectrum of pure MM. Signals of a minor amount of mismatched duplex
species appear only in the spectra of M4 and MS. This result agrees with the observed
behaviour in the D20 spectra of Ml to MS.
A comparison between the imino-proton spectra of the intact 12-mer duplex M3 (Figure
4E) and those of the nicked duplex structures with (Figure 4D) and without (Figure 4C)
the presence of a 5'-phosphorylated terminus at the interruption shows that the major shift
differences are observed in the spectral region 13.8-13.5 ppm. The three low-field iminoproton signals of M3 are assigned to the three A * T base pairs in the core of the duplex
on the basis of their resonance positions. The lowest-field inino-proton resonance is safely
identified with the T(9) A(16) base pair by comparison with the duplex structures of II
+ III (Figure 4A). The imino-proton signals that resonate between 13.8-13.5 ppm are
assigned to the neighbouring T A base pairs T(6) A(19) and A(7) T(18) in the duplex
structure. The latter two signals display upfield shift effects when a single-stranded break
is introduced. When the 3'-phosphodiester bond between T(18) and dA(19) is cleaved (AS)
one of these signals shifts remarkably upfield (Figure 4D). However, a single-stranded
interruption between these base pairs without a phosphate group at the nick causes
pronounced upfield shifts of both imino-proton resonances. These effects probably reflect
some conformational changes in the presence of a nick in the duplex. An increase of the
exchange rate between the imino protons (6,18) and the solvent alone can be discounted.
These observations correspond well to the results obtained from the chemical-shift
considerations of the non-exchangeable protons of Ml, M2 and M3. These investigations
show that the duplex is locally slighdy distorted in the prsence of a single-st break.
These distortions become more pronounced when the phosphate between T(18) and dA(9)
is removed.
Ligation
The relatively small system (M2), only a single helix turn, was investigated as a model
for a single-stranded break in native DNA. Therefore, an in vitro ligation experimnt was
undertaken.
For enzymatic activity the DNA ligase requires a free 3'-OH group of the DNA-chain
and a phosphate at the 5' end of the other strand. Furthermore, the DNA stands to be
joined by DNA ligase must be part of a double-helical molecule. The energy required
to form the phosphodiester linkage is provided by the cleavage of the ctj3-pyrophosphate
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1
2
3
4
5
6
7
8
9 10
11
12
Figure 5. Autoradiogram of a 20 % polyacrylamide gel after electrophoresis of the reaction products at various
time intervals. Aliquots of 10 ml were used for each slot.
(slot 1) 0 minutes; (2) 5 minutes; (3) 10 minutes; (4) 20 minutes; (5) 40 minutes; (6) 1 hour; (7) 2 hours; (8)
1.5 hours; (9) 3 hours; (10) 5 hours; (11) 24 hours; (12) d(pTGCAGTACGGCT) marker.
Note that for visualization of the progress of the reaction at increasing incubation time lanes 7 and 8 should
be interchanged.
bond of ATP (phage or eukaryotic enzymes) or by the cleavage of the pyrophosphate bond
of NAD (bacterial enzymes).
First, experimental conditions were explored under which the nicked duplex structure
(M2) could be formed completely at low oligonucleotide concentration (= 30 IM). It is
well known that Mg2+-ions affect the stability of tRNAs [30,31]. Heerschap [31] showed
that at increasing Mg2+-concentration the melting temperature reached a plateau value;
after addition of 10 mM Mg2+ or more the Tm value remains constant. The effect of
addition of Mg2 + upon the melting temperature of M2 was measured
spectrophotometrically at Mg2+-concentrations varying between 15 mM and 50 mM.
These experiments resulted in minor changes of the melting temperatures (26-29 °C).
This observation indicates that at the experimental Mg2e-concentrations the plateau Tm
value is already reached.
The ability of T4 ligase to join Ia + II was studied by means of incorporation of 32p
during the ligation of Ia* +. II. Assays were carried out at 14 °C in order to maintain
the duplex structure of the molecule. Polyacrylamide slab gel electrophoresis under
denaturating conditions was used as a direct method for visualization of the progress of
the reaction. Aliquots were taken from the reaction mixtures from 0 to 24 hours.
Figrre 5 shows an autoradiogram of the gel. After 40 minutes of incubation a second
welI-resolved band appears in the autoradiogram. A comparison of the migration position
of the new species with that of the 12-mer (IV) Oane 12) clearly demonstrates that the
species formed represents the expected dodecamer fragment. The other band corresponds
to the 32P-labeled hexamer (Ia*). When the incubation time is prolonged the amount of
joined species increases progressively. The reaction is almost completed after an overnight
incubation (lane 11).
From the high efficiency of joining of the two hexamer fragments by T4 ligase we suggest
that M2 is probably representative for single-stranded breaks in DNA in biological systems.
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Thus, it seems likely that the structural behaviour of oligonucleotides can be transferred
largely to conformational properties of native DNA.
CONCLUSIONS
The DNA compounds d(ACGGCT) (I), d(pACGGCT) (Ia), d(TGCAGT) (HI),
d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV) and equimolar mixtures thereof
were studied by means of NMR spectroscopy. It was shown that the equimolar mixtures
MI (I + II + III) and M2 (Ia + HI + III) adopt a B-DNA type duplex structure with
a nick in the core of the molecule at low temperature. A comparison between the chemicalshift data of the duplexed structure comprising of the complementary strands III + IV
(M3) and those of the nicked compounds demonstrates that the introduction of a singlestranded break results in small local distortions in the B-type duplex structures. The structural
changes appear more pronounced when the phosphate at the interruption is removed. The
largest shift changes are found in the base pairs that flank the nick.
Thermodynamic analysis of Ml yields an average Tmd value of 359 K (1 M DNA) and
AHIL of -312 kJ * mol- I for the duplex/coil transition. For this equilibrium of M3 a Tmd
value of 361 K (1 M DNA) and a AH3 value of -356 kJ mol-I are calculated. It is
concluded that cooperativity of the hexamer strands occurs in the melting behaviour of Ml.
The ligation experiment of M2 with T4 polynucleotide ligase clearly demonstrates that
the small substrate is efficiently joined.
ACKNOWLEDGEMENTS
This research was supported by the Netherlands Foundation for Chemical Research (SON)
with financial aid from the Netherlands Organization for Scientific Research (NWO). NMR
spectra were recorded at the Dutch National 600-200 MHz NMR Facility at Nijmegen
and on the 300 MHz spectrometer in the Department of Chemistry at Leiden. The
biochemical experiments were carried out at the Department of Biochemistry at Leiden.
We wish to thank Mr. P.A.W. van Dael, Mr. C. Erkelens and Mr. J.J.M. Joordens for
technical assistance.
*To whom correspondence should be addressed
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Nucleic Acids Research
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