Tài liệu Báo cáo Y học: Dynamic mechanism of nick recognition by DNA ligase ppt

REVIEW ARTICLE
Dynamic mechanism of nick recognition by DNA ligase
Alexei V. Cherepanov* and Simon de Vries
Kluyver Department of Biotechnology, Delft University of Technology, Delft, the Netherlands
DNA ligases are the enzymes responsible for the repair of
single-stranded and double-stranded nicks in dsDNA. DNA
ligases are structurally similar, possibly sharing a common
molecular mechanism of nick recognition and ligation
catalysis. This mechanism remains unclear, in part because
the structure of ligase in complex with dsDNA has yet to be
solved. DNA ligases share common structural elements with
DNA polymerases, which have been cocrystallized with
dsDNA. Based on the observed DNA polymerase–dsDNA
interactions, we propose a mechanism for recognition of a
single-stranded nick by DNA ligase. According to this
mechanism, ligase induces a 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 dissociation
of the enzyme. For a nicked dsDNA substrate, the con-
traction of the enzyme-bound DNA motif (a) triggers an
opened–closed conformational change of the enzyme, and
(b) forces the motif to accommodate the strained A/B-form
hybrid conformation, in which the nicked strand tends to
retain a B-type helix, while the non-nicked strand tends to
form a shortened A-type helix. We propose that this con-
formation is the catalytically competent transition state,
which leads to the formation of the DNA–AMP interme-
diate and to the subsequent sealing of the nick.
Keywords: DNA ligase; nick recognition; A-form DNA;
A/ B-form DNA hybrid; protein–DNA interactions; B-A

DNA helix transition.
DNA ligases are the enzymes that catalyze the joining of
single- and double-stranded nicks in dsDNA [1]. These
enzymes play a pivotal role in replication, sealing the nicks
in the lagging DNA strand [2–5]. They also participate in
DNA excision [6–8], double-strand break repair [9–12] and
take part in DNA recombination [10,13–15]. The mechan-
ism of enzyme catalysis (Scheme 1) includes three main
steps: (1) covalent binding of the nucleoside monophos-
phate, AMP or GMP, via the e-amino lysyl phosphorami-
date bond, (2) transfer of the nucleotidyl moiety onto the
5¢-phosphate end of the nick, forming an inverted pyro-
phosphate bridging structure, A(G)ppN and (3) formation
of the phosphodiester bond between the 3¢-OH and the
5¢-phosphate ends of the nick, releasing the nucleotide.
Scheme 1. Mechanism of the ATP-dependent
end-joining activity of T4 DNA ligase. nds-
DNA, dsDNA containing a 5¢-phosphorylated
nick. n-MgAMP-dsDNA, nicked dsDNA
adenylylated at the 5¢-phosphate of the nick.
Correspondence to S. de Vries, Kluyver Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft,
the Netherlands Tel.: + 31 15 2785139, Fax: + 31 15 2782355,
E-mail:
Abbreviations: EMSA, electrophoretic mobility shift assay.
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 8 July 2002, accepted 11 October 2002)
Eur. J. Biochem. 269, 5993–5999 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03309.x
STRUCTURE OF DNA LIGASES

The crystal structures of several ATP- and NAD
+
-depend-
ent DNA ligases have been solved: the bacteriophage T7
DNA ligase complex with ATP [17,18], the enzyme–AMP
covalent complexes of the eukaryotic DNA ligase from
Chlorella virus [19] and of the thermophilic bacterium
Thermus filiformis [20,21]. In addition, the structure of the
adenylylation domain of the NAD
+
-dependent DNA ligase
from Bacillus stearothermophilus has been determined [22].
Analyses indicate that these proteins are very similar [23,24],
and that the minimal catalytic core of the ATP-dependent
DNA ligase consists of two structurally conserved domains
(Fig. 1). The N-terminal domain 1 (blue and green regions,
Fig. 1) contains the active site, where the adenylylation of
the enzyme takes place. Within domain 1, a smaller
subdomain (1c) can be distinguished (36–159 for T7 DNA
ligase and 30–104 for the Chlorella virus DNA ligase, Fig. 1,
shown in blue), which contains a mobile loop, invisible in
the crystal structure. Domain 1 contains four spatially
conserved positively charged residues (Fig. 1, blue) that are
proposed to interact with the 5¢-phosphate moiety of the
nick [19,25]. Two of them, Lys238 and Lys240 of T7 DNA
ligase (Lys188 and to a lesser extent Lys186 of Chlorella
virus DNA ligase) were shown to be essential for the
transadenylation and nick sealing activities [25,26]. Lys240
forms a photo-crosslinking adduct with the 5¢-terminal
nucleotide of the nick, implying its direct involvement in

binding of the nick phosphate [25]. Domain 1 contains the
catalytic lysine residue that forms a covalent intermediate
with the nucleotide coenzyme.
The C-terminal domain of DNA ligase, domain 2 (Fig. 1,
yellow), is smaller and is connected to domain 1 via the
conserved motif D [25] (Fig. 1, red, in alternative notation
called as motif V [27]). Domain 2 is also referred to as the
OB (oligonucleotide/oligosaccharide binding)-fold domain,
similar to those found in other DNA and RNA binding
proteins [28,29]. Domain 2 is flexible; it was shown for the
related nucleotidyltransferase, the mRNA capping enzyme
from Chlorella virus PBCV-1, that during catalysis the
enzyme undergoes opened–closed conformational changes
upon which domain 2 moves towards domain 1 and closes
the nucleotide binding site [30]. For DNA ligases it was
suggested that this motion is connected with binding of both
ATP and nicked dsDNA [25]. As to ATP, closing of domain
2 was proposed to adjust the conformation of the b–c
pyrophosphate of ATP to a position favorable for the
in-line nucleophilic attack of the catalytic lysyl moiety
[19,23]. As to nicked dsDNA, closing of the domain 2 was
proposed to clamp the enzyme on DNA [25].
dsDNA BINDING SITE
Studies involving limited proteolysis, mutagenesis and
molecular modeling strongly suggest that dsDNA binds
ligase in the cleft between domains 1 and 2 [17,21,25,31–33].
Both domains of T7 DNA ligase bind dsDNA independ-
ently, and, as expected, only domain 1 retains residual ligase
activity [33]. On the basis of modeling studies it was shown
that the motifs A and B of subdomain 1c, and C and D of

domain 1 (Fig. 1, red) are involved in dsDNA binding [25].
It was suggested that the dsDNAÆprotein contacts traverse
the whole of domain 1, and that the dsDNA binds right on
top of the AMP bound in the active site [25,34]. The
modeling did not elucidate a possible dsDNA-binding site
of domain 2, perhaps because the opened conformation of
the enzyme was used. Using DNA footprinting analysis it
was shown that ligase binds nicked dsDNA asymmetrically,
contacting 7–12 nucleotides at the 5¢-phosphate side of the
nick, and 3–8 nucleotides at the 3¢-hydroxyl side [25,32,34].
With respect to the enzyme structure that would mean that
motifs A and B must contact the 5¢-phosphate side of the
nick of the dsDNA, because they are further away from the
active site compared to motifs C and D [25].
STRUCTURAL SIMILARITY BETWEEN
DNA LIGASE AND DNA POLYMERASE
The catalytic core of DNA polymerase responsible for the
dsDNA elongation activity contains three domains. Its
shape resembles a half-opened hand (Fig. 2, left), and the
domains are named accordingly [35,36]. The catalytic ÔpalmÕ
domain contains the polymerase active site, where the
incorporation of the nucleotide in the nascent primer chain
takes place. dsDNA binds the palm domain in the cleft
formed by the ÔthumbÕ and flexible ÔfingersÕ [37]. Similar to
DNA ligase, DNA polymerase undergoes an opened–closed
conformational change in the course of catalysis, upon
which the fingers and the thumb domains close on the palm
domain containing bound dsDNA and dNTP [37–41].
Fig. 1. Structure of T7 DNA ligase (left) and
the DNA ligase–adenylylate complex from the

Chlorella virus (right). Domain 1 is shown in
green, subdomain 1c in blue and domain 2 in
yellow. Motifs A–D are shown in red. Resi-
dues that participate in binding of the nick
phosphate are shown in blue. Some hydro-
phobic residues in the putative DNA binding
siteareshowninred.TheAMPmoietyof
Chlorella virus DNA ligase adenylate is shown
in purple.
5994 A. V. Cherepanov and S. de Vries (Eur. J. Biochem. 269) Ó FEBS 2002
Figure 2 shows that the analogy with a hand can be
extended to DNA ligase. Domain 1 would be associated
with the palm, subdomain 1c with the thumb and domain 2
with the flexible fingers. Also, the dsDNA binding mode
proposed for DNA ligase in the modeling studies [25].
resembles the one of DNA polymerase (Fig. 2, left).
dsDNA–POLYMERASE AND
dsDNA–LIGASE INTERACTIONS
The interaction of DNA polymerase with dsDNA is
relatively well understood. In solution dsDNA generally
prefers the B-type helix, but in the complex with polymerase
up to 6 or 7 bp at the 3¢-end of the primer accommodate the
A-form [41–43] (Fig. 2, left). One of the reasons for this is
that the polymerase bends DNA, clamping the helix
between the palm and the thumb domains [44–48]. A–B-
form dsDNA hybrids are usually bent at the junction
[49,50], so the induced bending by the protein stabilizes the
A-form [42,51,52]. Another reason for the relative stability
of A-form dsDNA in complex with the DNA polymerase is
relatedtolessspecificdsDNAÆprotein interactions. They

include (a) relatively high hydrophobicity of the dsDNA
binding cleft compared to solution, which leads to a
decrease of the degree of hydration of bound dsDNA,
which stabilizes the A-type helix [53,54], and (b) replacement
and/or exclusion of water molecules, which are normally
hydrogen bonded to the dsDNA in solution, by the amino
acid residues [55–57] and/or salt bridges [42] in the
dsDNAÆprotein complex. The resulting effect can be
compared with the addition of a hydrophobic solvent or
with an increase of the ionic strength, factors which induce
the B-to-A helix transition of dsDNA in solution [58,59]. In
general, the induced B-to-A helix transition is a common
feature of dsDNAÆprotein interactions [52,60–62], in par-
ticular for the enzymes that catalyze sealing/cutting oper-
ations on dsDNA [42].
It seems likely that the A-B dsDNA hybrid bend at the
junction would fit DNA ligase better than the straight
dsDNA, because the cleft between domains 1 and 2 is
curved. In this case motif A of subdomain 1c (thumb) would
contact the hybrid dsDNA at the A-B junction point,
similar to the thumb–helix clamp motif of HIV-1 RT
(Fig. 2, left). The distance between the junction point and
the nick binding site is around 20 A
˚
, which corresponds to
 7 bp of dsDNA. There are several aromatic residues in
the active site of DNA ligase, which could stabilize the
A-helix by hydrophobic and/or aromatic–aromatic interac-
tions. Surprisingly, most of them are aligned parallel to each
other along the putative dsDNA binding site (Fig. 1, red).

DNA ligase undergoes an opened–closed conformational
change during catalysis, which could stabilize the A-DNA
motif by water exclusion and additional protein–DNA
interactions. The pyrophosphate-bridging riboadenosine at
the 5¢-end of the dsDNA nick might stimulate the B-to-A
DNA conversion as well because of the structural influence
of ribose sugar, similar to the other cases of A-DNA
duplexes that contain a single ribose residue [63–65]. If the
7 bp-long fragment of bound dsDNA would adopt the
A-form conformation in DNA ligase complex, it would
cause an overall DNA unwinding of  20–25 degrees,
because A-DNA contains roughly one more bp per turn of
the helix than the B-form. It was shown that both ATP- and
NAD
+
-dependent DNA ligases unwind dsDNA at the
binding site at least for 17–20 degrees per bound molecule of
theenzyme[66].
Therefore, there are sufficient grounds to suggest that the
6–9 bp-long B-DNA, at the 5¢-phosphate side of the nick,
changes to A-DNA after formation of the DNA–ligase
complex, similarly to the motif at the 3¢-OH primer end of
the dsDNA bound to DNA polymerase. What could be the
role of this transition in the DNA ligase catalysis?
DYNAMIC MECHANISM OF NICK
RECOGNITION BY DNA LIGASE –
AHYPOTHESIS
According to Doherty et al. [25,34], the adenylylated DNA
ligase binds dsDNA forming nonspecific contacts with
motifs A, B, C and D. According to our hypothesis the

enzyme, in addition, bends dsDNA at the point of contact
with motif A. Subdomain 1c, similar to the thumb domain
of DNA polymerase, clamps on dsDNA bound in the
crevice formed by domain 1 (palm) and domain 2 (fingers).
This could be achieved by moving the tip (motif B) of
subdomain 1c (thumb) towards domains 1 (palm), 2
(fingers) and bound dsDNA (Fig. 2, right, white arrow),
similar to the motion of the thumb domain in DNA
polymerases [35,37,41]. Nonspecific interactions lead to a
decrease of the degree of hydration of the bound DNA. As a
Fig. 2. Structures of DNA polymerase domain
of HIV-1 reverse transcriptase in complex with
dsDNA (left), and T7 DNA ligase (right). The
connection domain of HIV-1 RT is omitted
from the figure for clarity. The palm domain is
showningreen,thethumbdomaininblueand
the fingers domain in yellow. Directions of the
catalytic movement of the thumb and fingers
domains are indicated with white arrows.
Ó FEBS 2002 Nick recognition by DNA ligase (Eur. J. Biochem. 269) 5995
result, the 6–9 bp dsDNA fragment between motifs A and
C changes to the A-form helix. This transition is accom-
panied by a dsDNA contraction of 5–7 A
˚
,becausethe
distance between the neighboring nucleotides is 2.6 A
˚
in the
A-form vs. 3.4 A
˚

in the B-form. Contraction causes dsDNA
to slip in the active site towards the clamp site (motif A)
(Fig. 3). It also causes an overall DNA unwinding for 20–30
degrees (6 bp A-DNA contains  0.54 bp more per turn of
a helix compared to 6 bp B-DNA, which corresponds to
360 degrees · 0.54/10–20 degrees unwinding angle).
It is known that the 5¢-nick phosphate is essential for the
tight binding of dsDNA by the DNA ligase. Several
residues (Fig. 1, blue), which are located in domain 1 close
to the bound AMP, are thought to bind to this moiety
[19,25]. We propose that DNA ligase makes a two-force-
point contact with nicked dsDNA – at the clamp site via
motifAandatthe5¢-phosphate of the nick via the specific
phosphate-binding residue(s) (e.g. Lys238 and Lys240 for
T7 DNA ligase [19,25], or Arg42, Arg176 and Lys186 for
Chlorella virus ligase [19,26]). At the clamp site, both
DNA strands are fixed with respect to the enzyme, because
DNA bends here. At the nick, however, only the
5¢-phosphate of the nicked strand is enzyme-bound
(Fig. 3, ds nicked DNA). During the contraction, or
B-to-A DNA helix transition, the non-nicked strand tends
to adopt the A-DNA conformation, because it is anchored
to the enzyme only at the clamp site and is free to slip in
the active site.
According to our hypothesis, the nicked strand has less
freedom of conformational changes because it is anchored
to DNA ligase at two points. Two extreme cases can be
considered. The first case represents an enzyme, which
would be structurally infinitely flexible between the two
force points. In this case, contraction of dsDNA would drag

the residues bound to the 5¢-phosphate of the nick several
angstroms towards motif A. Some of these residues (e.g.
Lys238 and Lys240 for T7 DNA ligase or Lys186 and
Lys188 for Chlorella virus ligase) belong to motif D. This
motif connects domains 1 and 2, and serves as a ÔhingeÕ
during the opened–closed conformational change. So, the
nick phosphate of dsDNA could pull on this hinge during
contraction, triggering the closing of domain 2, and could
further stall the ligase in the closed conformation until the
nick is sealed.
The other extreme case would be that the enzyme is
structurally infinitely rigid between motif A and the nick
phosphate-binding residue(s). In this case, the nicked strand
wouldtendtoretainitsB-form,sinceitisfixedbothatthe
clamp site and at the 5¢-phosphate of the nick. As a result,
the DNA motif between the clamp site and the nick
phosphate would adopt a strained hybrid conformation, in
which the non-nicked strand is more A-like, while the
nicked strand is more B-like (Fig. 3, nicked dsDNA, closed
enzyme). One of the options for DNA to retain the
hydrogen bonding of the 3¢-terminal base pair of the nick
would be to slightly rotate counterclockwise around the
helical axis, so that the 3¢-OH moiety would move towards
the 5¢-phosphate of the nick and forward in the 3¢-direction
of the nicked strand (Fig. 3, nicked dsDNA, closed
enzyme). In other words, the 5¢-phosphate would move
towards the protein interface, while the 3¢-OH group would
move towards the solution. In this way, the 3¢-OH group
would adopt the apical configuration in respect to the
a-phosphorus moiety of the AMP cofactor (Fig. 4). For

comparison, the nicked dsDNA bound to the DNA
polymerase b makes a similar motion (for structure cf
[39]), only in that case the 3¢-OH side of the nick moves
away from the 5¢-phosphate of the nick and backwards in
the 5¢-direction of the nicked strand.
Fig. 3. Illustration of the proposed mechanism
of dynamic nick recognition by DNA ligase.
(Left) binding of the dsDNA. (Right) binding
of the nicked dsDNA. B-form DNA is colored
yellow, A-DNA is blue and the enzyme is
showningreen.
Fig. 4. Illustration of the proposed mechanism of dynamic nick recog-
nition by DNA ligase. Positioning of the reacting groups in the active
site of the enzyme for the B-DNA configuration and for the A–B
strained DNA hybrid.
5996 A. V. Cherepanov and S. de Vries (Eur. J. Biochem. 269) Ó FEBS 2002
Most probably, the flexibility of DNA ligase is somewhere
between these two extreme cases, so that the B-to-A helix
transition of dsDNA would cause both the closure of domain
2 and the formation of the strained A–B configuration.
In summary, if our hypothesis of dynamic nick recog-
nition proves to be correct, DNA ligase would be a good
example of an enzyme that acts according to induced-fit
and strain mechanisms of catalysis in which both the
enzyme and the substrate undergo significant conforma-
tional changes to achieve the transition state configuration
[67–69].
BINDING OF dsDNA TO DNA LIGASES
It is important to note that the binding of dsDNA to the
ligase is still a matter of some controversy. The results based

on the electrophoretic mobility shift assay (EMSA) indicate
that the ligase does not bind the non-nicked dsDNA, or
dsDNA containing the nonphosphorylated nick [25,32,70].
On the other hand, other experiments that show relaxation
of supercoiled DNA in the presence of T4 DNA ligase imply
that the enzyme not only binds but also unwinds the non-
nicked DNA helix [66]. In our opinion, the reason for this
paradox is that the EMSA fails to detect proteinDNA
complexes with k
off
values comparable to the apparent rate
constant for diffusion of the proteinDNA complex through
the pore of the acrylamide gel, k
diff
app
.Fora5%gelthe
apparent pore diameter is around 100–200 nm, depending
on the bisacrylamide content [71]. Thus, for a 2 h separation
with an electrophoretic shift of, for example, 5 cm, the k
diff
app
can be estimated as 5 · 10
)2
m/200 · 10
)9
m ¼ 2.5 ·
10
5
pores per 2 h, or 35–70 s
)1

. This implies that only the
complexes with k
off
of about 1–2 s
)1
would be detected
using this method. On the other hand, more rapidly
exchanging complexes can be detected in the assay showing
relaxation of the supercoiled DNA.
B-TO-A DNA HELIX TRANSITION – A
DYNAMICTESTOFTHESTATEOFTHE
DNA SUBSTRATE
We propose that the B-to-A DNA helix transition serves as
a dynamic test to determine the state of the DNA substrate,
and is used by DNA ligase to comply with its fidelity
requirements.
(A) To test for the presence of mismatching nucleotide(s)
at the 3¢-hydroxyl side of the nick. Even though the A-B
strained conformation can be adopted, the dangling 3¢-OH
end would not occupy the position apical towards the
leaving AMP, preventing the sealing of the nick, and,
possibly, hindering the preceding transadenylation. This
agreeswiththefactthatmismatchesatthe3¢-OH side of the
nick in some cases inhibit not only the nick sealing activity
[72], but adenylylation as well [73].
(B) To lower the large sequence-dependent structural
variations at the 5¢-PO
4
side of the nick, and to test for the
presence of mismatching nucleotide(s). The A-form of DNA

is known to obey Ôstructural conservatismÕ, being rather
independent of the primary sequence [74]. The presence of
mismatches at the 5¢-PO
4
side of the nick destabilizes the
A-form helix increasing the DNA hydration, because the
water molecules tend to cluster around unusual base pairs to
compensate for the absent hydrogen bonds [75].
(C) To test for the presence of an RNA motif at the 5¢-end
of the nick. This important fidelity requirement would
preclude DNA ligase to join the Okazaki fragments that
contain RNA primer fragments, before they are removed by
the 5¢)3¢-exonuclease activity of DNA polymerase [76] or
by the action of specific RNases [77]. The B-to-A helix
transition would not occur in case of the nick containing
5¢-RNAÆDNA, because the RNAÆDNA hybrid already
adopts the A-like form in solution. As a result, the A-B
strained conformation would not be achieved, the 3¢-OH
group of the nick would not occupy the position apical to
the leaving AMP and the nick sealing would be inhibited.
The latter agrees with the fact that the DNA ligase joins
5¢-RNAÆDNA to the 3¢-DNAÆDNA poorly, leading to the
accumulation of the DNA-adenylate intermediate, while the
opposite situation results in effective ligation [78,79]. It is
necessary to note, however, that in certain cases DNA ligase
is capable of joining nicks containing the RNA/DNA motif
on the 5¢-side with reduced efficiency [72,79–82]. In these
cases, generally, oligo-d(r)A/oligo-r(d)T sequences were
ligated, which, for dsDNA, have a very low tendency to
form the A-helix in solution [83–85]. The A-helix is not the

only possible conformation of the DNAÆRNA chimera;
sometimes it rather adopts a mixed A–B-geometry [86,87],
or, under certain conditions, even the B-helical conforma-
tion [88]. Therefore, it is possible that the oligo-d(r)A/oligo-
r(d)T DNAÆRNA hybrids in solution adopt the B-like
structure, and in complex with DNA ligase undergo a
B-to-A helix transition, allowing nick-joining.
In summary, we propose that DNA ligase transiently
probes dsDNA by bending the DNA helix, unwinding, and
inducing the B-to-A helix transition. A defect in the DNA
helix, such as a phosphorylated nick reveals itself during the
dynamic test, forcing (a) DNA ligase to form a stable
complex with dsDNA by changing to a closed conforma-
tion, and (b) dsDNA to adopt a conformation favorable for
the transadenylation and sealing of the nick.
ACKNOWLEDGEMENTS
This work was supported by Association Of Biotechnology Centers In
the Netherlands (ABON) (Project I.2.8) and by the Netherlands
Research Council for Chemical Sciences (CW) with financial aid from
the Netherlands Technology Foundation (STW) (grant 349–3565).
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