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

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

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

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).

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
˚

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

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

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

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