Báo cáo khoa học: 3¢- to 5¢ DNA unwinding by TIP49b proteins docx

3¢-to5¢ DNA unwinding by TIP49b proteins
Christophe Papin
1,2,
*
,
, Odile Humbert
1,2,
*, Anna Kalashnikova
1,2
, Kelvin Eckert
3
, Solange Morera
3
,
Emmanuel Ka
¨
s
1,2
and Mikhail Grigoriev
1,2
1 Laboratoire de Biologie Mole
´
culaire Eucaryote, Universite
´
de Toulouse, France
2 CNRS, LBME, Toulouse, France
3 Laboratoire d’Enzymologie et Biochimie Structurales, Gif-sur-Yvette, France
Introduction
The highly conserved TIP49a and TIP49b proteins
(also called pontin and reptin; a complete list of names
is provided elsewhere [1]) belong to the AAA

+
super-
family of P-loop ATPases (i.e. ATPases associated
with diverse cellular activities), which includes enzymes
involved in cellular housekeeping, cell division and dif-
ferentiation. This superfamily is composed of a broad
variety of enzymes, which appear to have a common
property: energy-dependent remodeling of proteins
and ⁄ or nucleic acids that results in the unfolding and
disassembly of target macromolecules. The AAA
+
proteins contain conserved ATP-binding and hydroly-
sis domains, which are activated by the formation of
oligomeric assemblies. ATP binding and hydrolysis
induce the conformational changes in the protein that
are required for the translocation or remodeling activi-
ties of target substrates [2–4].
TIP49a and TIP49b are implicated in a variety of cel-
lular processes, such as chromatin remodeling during
double strand-break repair, transcriptional regulation,
genome stability, small nucleolar RNA biogenesis, telo-
merase holoenzyme assembly and cellular division dur-
ing mitosis [1,5,6]. Being essential proteins in yeast and
embryonic-lethal in higher eukaryotes, they show a com-
plex network of protein–protein interactions where, in
some cases, they play antagonistic roles during the
Keywords
AAA+ ATPases; DNA binding; reptin; Rvb2p;
TIP49b
Correspondence

E. Ka
¨
s or M. Grigoriev, IBCG, 118 route de
Narbonne, 31062 Toulouse Cedex 9, France
Fax: +33 (0)5 61 33 58 86
Tel: +33 (0)5 61 33 59 19;
+33 (0)5 61 33 59 59
E-mail: ;

*These authors contributed equally to this
work
Present address
IGBMC UMR 7104, Illkirch, France
(Received 4 January 2010, revised 9 April
2010, accepted 14 April 2010)
doi:10.1111/j.1742-4658.2010.07687.x
TIP49b (reptin) is an essential eukaryotic AAA+ ATPase involved in a
variety of cellular processes, such as chromatin remodeling during double-
strand break repair, transcriptional regulation, control of cell proliferation
and small nucleolar RNA biogenesis. How it acts at the molecular level
remains largely unknown. In the present study, we show that both human
TIP49b and its yeast orthologue, Rvb2p, cooperatively bind single-stranded
DNA as monomers. Binding stimulates a slow ATPase activity and sup-
ports a 3¢-to5¢ DNA unwinding activity that requires a 3¢-protruding tail
‡ 30 nucleotides. The data obtained indicate that DNA unwinding of 3¢-to
5¢ junctions is also constrained by the length of flanking duplex DNA. By
contrast, TIP49b hexamers were found to be inactive for ATP hydrolysis
and DNA unwinding, suggesting that, in cells, protein factors that remain
unknown might be required to recycle these into an active form.
Structured digital abstract

l
MINT-7804328: TIP49b (uniprotkb:Q4QQS4)andTIP49b (uniprotkb:Q4QQS4) bind (MI:0407)
by electron microscopy (
MI:0040)
l
MINT-7804638: tip49b (uniprotkb:Q4QQS4) and tip49b (uniprotkb:Q4QQS4) bind (MI:0407)
by molecular sieving (
MI:0071)
Abbreviation
ssDNA, single-stranded DNA.
FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS 2705
transcriptional regulation of gene expression and embry-
onic development. However, their molecular mecha-
nisms of action remain to be elucidated, particularly in
view of such diverse functions.
Although ATP hydrolysis is most likely essential for
the functions of these proteins [7,8], most biochemical
analyses of TIP49 proteins have failed to detect
significant ATPase activity of the purified recombinant
proteins [9–12]. Some studies have indicated helicase
activity of TIP49 proteins, although opposite direction-
alities of DNA unwinding have been reported [13–15].
However, as in the case of ATP hydrolysis, little pro-
gress has been made with respect to further elucidating
the DNA processing activities of TIP49 proteins or
establishing whether their wide range of cellular func-
tions is related, if at all, to transactions based on
DNA unwinding.
Because TIP49 proteins are found in chromatin
remodeling complexes such as TIP60, Ino80 and

SWR1 and are necessary for their activity, it is plausi-
ble that they might partake in protein ⁄ DNA interac-
tions that play a role in functions such as DNA repair
or transcriptional control, in addition to the scaffold-
ing role attributed to them in the formation of multi-
protein complexes [1]. In the present study, we
demonstrate that TIP49b and its yeast orthologue
Rvb2p are indeed DNA-binding proteins. Cooperative
binding to single-stranded DNA (ssDNA) stimulates a
weak ATPase activity, which in turn leads to subse-
quent DNA unwinding off 3¢-protruding tails ‡ 30
nucleotides in length. We show that these properties,
(i.e. ssDNA-dependent weak ATPase and slow 3¢-to
5¢ unwinding) have been conserved between the mam-
malian and yeast TIP49b proteins.
Results
To examine the biochemical properties of human and
yeast reptin (TIP49b and Rvb2p, respectively), we
purified them from Escherichia coli as N-terminal
FLAG ⁄ His
6
- and C-terminal His
6
-tagged forms,
respectively. The gel filtration profile on Superdex
S-200 revealed that TIP49b is eluted in three well-sepa-
rated fractions (Fig. 1A): the high molecular-weight
peak, containing TIP49b aggregates (not used in the
present study) and two other peaks eluting in the
ranges 440–158 and 67–43 kDa, respectively. Native

protein gel electrophoresis (the inset in Fig. 1A shows
a silver-stained gel) and electron microscopy analysis
(Fig. 1B) of these fractions confirmed that they differ
in their oligomeric state: fraction 1 was found to be
mostly hexamers (Fig. 1B, left), whereas fraction 2
mostly contained TIP49b monomers (Fig. 1B, right).
SDS ⁄ PAGE analysis of aliquots of the protein prepa-
rations followed by silver staining and western blotting
using aFLAG or aTIP49b sera revealed the presence
MonomerHexamer
TIP49b:
020
100 nm 100 nm
40 60 80 100
mL
mAU
20
A
B
CD
15
10
5
0
2000 158 67 43 kDa
440
TIP49b
RecA
–+
+

+
123
ssM13
antiFLAG
antiTIP49b
kDa
12
55
36
31
21
66
97
Western blottingSilver staining
Hexamer
Monomer
Fig. 1. TIP49b proteins used in the present study. (A) Gel filtration
profile of TIP49b on a Superdex S-200 column. The fractions further
analyzed are indicated by the horizontal bars. Silver-stained aliquots
of each fraction analyzed by native PAGE are shown in the inset.
(B) Electron microscopy images of TIP49b purified as hexamers
(left) and monomers (right) used in the present study. Magnifica-
tion: · 220 000; scale bar = 100 nm. (C) Purified proteins (500 ng)
were analyzed by SDS ⁄ PAGE on 4–20% Tris–glycine SDS gels
followed by silver staining and western blotting using aFLAG
(Sigma) or aTIP49b (BD Biosciences) sera as indicated. Black
arrowheads indicate TIP49b doublets; the grey arrowhead shows
the minor GLMS_ECOLI contaminant. (D) UV cross-linking in the
presence of radiolabeled [cP
32

]-ATP followed by SDS ⁄ PAGE per-
formed with RecA (lane 1) or with TIP49b in the absence (lane 2)
or in the presence (lane 3) of ssM13. Black arrowheads show the
TIP49b doublets (lanes 2 and 3); grey arrowheads indicate minor
species that most likely correspond to UV-crosslinked RecA (lane 1)
or TIP49b (lanes 2 and 3) intramolecular species.
DNA unwinding by TIP49b C. Papin et al.
2706 FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS
of purified TIP49b monomers (Fig. 1C), which appear
as doublets (black arrowheads) suitable for further bio-
chemical analysis. The major contamination detected
by LC-MS ⁄ MS was a 67 kDa GLMS_ECOLI protein
(Fig. 1C, grey arrowhead). We did not detect contami-
nation by bacterial ATPases and helicases in our
TIP49b and Rvb2p protein preparations.
UV cross-linking in the presence of radiolabeled
ATP was performed next (Fig. 1D). RecA (33 kDa)
(5 lm), used as a positive control (lane 1), or TIP49b
(lanes 2 and 3) were incubated in the presence of
[c-P
32
]-ATP and ssM13 at 50 ngÆlL
)1
, UV-crosslinked
and analyzed by SDS ⁄ PAGE on Tris–glycine gels.
Although TIP49b (black arrowheads) showed a lower
efficiency of ATP binding compared to that of RecA,
regardless of the presence of ssDNA, these results sug-
gest that the monomeric TIP49b preparations used
throughout the present study bind ATP and are not

detectably contaminated by other ATP-binding pro-
teins (Fig. 1D), except for very weak bands visible in
both RecA and TIP49b preparations that most likely
correspond to UV-crosslinked intramolecular species
(grey arrowheads).
ATPase activity of TIP49b and DNA binding
properties
ATP hydrolysis to ADP and inorganic phosphate by
TIP49b was assayed by incubating 1 lm of the protein
with 200 lm ATP containing [a-P
32
]-ATP tracer in the
presence of different nucleic acids (10 ngÆlL
)1
) and sep-
aration of reaction products by TLC (Fig. 2A). A small
accumulation of ADP above background was detected
in the presence of the protein alone, indicating intrinsic
ATPase activity (Fig. 2A, lane 2). As expected [13], the
ATPase activity was stimulated by single-stranded M13
DNA (ssM13, lane 4), but not by total RNA or circular
supercoiled pBR322 plasmid DNA (Fig. 2A, lanes 3
and 5). The steady-state ATPase activity of human
TIP49b and yeast Rvb2p (not shown) in the presence of
ssM13 was also measured in parallel: both proteins
obeyed Michaelis–Menten kinetics within the range of
0–2 mm ATP concentrations and showed similar kinetic
parameters, classifying both proteins as weak ATPases.
The hexameric fraction of TIP49b was also assayed and
found to be inactive for ATP hydrolysis under the same

experimental conditions (Fig. 2B). This suggests that
oligomerization of the protein might influence its enzy-
matic activity and DNA-binding properties (see below).
We next tested the effect of single-stranded oligonu-
cleotides on ATP hydrolysis by TIP49b compared to
that in the presence of ssM13 DNA (Fig. 2C). These
experiments were performed with 1 lm TIP49b,
200 lm ATP and saturating concentrations of synthetic
oligonucleotides of different lengths (21, 50, 80 and
115 mers; 10 ngÆlL
)1
). Stimulation of ATP hydrolysis
by TIP49b was found to be strongly dependent on oli-
gonucleotide length, with a plateau of approximately
75% and 45% for ssM13 and ss115, respectively, indi-
cating a correlation between ATPase activity and
ssDNA-binding properties. Accordingly, we measured
these as a function of protein concentration in gel-shift
experiments using synthetic oligonucleotides as probes
(Fig. 2D). In the titration shown, TIP49b retarded the
migration of ss115 (Fig. 2E, lanes 1–4) in a concentra-
tion-dependent manner but did not show significant
binding to ss21, whereas binding to ss85 was detected
at intermediate protein concentrations. The binding
curve of yeast Rvb2p to ss115 was comparable to that
of TIP49b, indicating the similar affinities of these con-
served proteins for ssDNA (data not shown).
It is significant to note that the length dependence
of ATP hydrolysis (Fig. 2C) parallels that seen for
DNA binding (Fig. 2D). Taken together, these results

suggest that physical interaction with ssDNA might be
directly involved in regulating the ATPase activity of
TIP49b. We also tested binding to duplex DNA under
the same conditions, demonstrating detectable yet
weaker binding with a similar concentration response
(Fig. 2E, lanes 5–8). Note that these experiments detect
distinct strong (black arrowheads) and weak (grey
arrowheads) complexes. At present, the nature of these
different complexes remains unknown, although we
speculate that they might correspond to species of dif-
ferent conformations preserved by native gel electro-
phoresis and ⁄ or to different stoichiometries of TIP49b
proteins bound to ss- or ds115 substrates. In the latter
case, the requirement for excess protein over DNA in
electrophoretic mobility-shift assays renders any esti-
mate of the number of TIP49b molecules bound to
ss- or ds115 necessarily speculative at best.
DNA unwinding by TIP49b and Rvb2p
The putative helicase activity of TIP49b is controverted
in the literature [9,13,15,16]. We addressed this issue by
measuring the ability of TIP49b monomers to unwind
short DNA duplexes in our experimental system
(Fig. 3). Using ss115-mer as a template, we prepared
5¢- and 3¢-ss ⁄ ds junctions (ss94ds21, containing a 21 bp
duplex region and a 94-nucleotide single-stranded tail;
Fig. 3A) to detect DNA unwinding activity, as well as
to investigate the direction of unwinding and the influ-
ence of different adenosine phosphate cofactors on this
process. The substrates were pre-incubated on ice with
protein for 15 min before the addition of cofactors.

C. Papin et al. DNA unwinding by TIP49b
FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS 2707
Reactions were allowed to proceed at 37 °C for 30 min
in the presence of a ten-fold excess of a trap
oligonucleotide, complementary to the unwound radio-
labeled probe, and then stopped by deproteinization in
a solution containing a 100-fold trap excess before
analysis by native gel electrophoresis.
The results presented in Fig. 3 demonstrate that
TIP49b indeed possesses a DNA-unwinding activity.
However, under our experimental conditions,
displacement of the labeled oligonucleotide is seen only
in the presence of ATP and has a strict specificity for
3¢- rather than for 5¢-ss ⁄ ds DNA junctions (Fig. 3A,
left, lanes 1–9). Yeast Rvb2p showed identical proper-
ties (Fig. 3A, center panel, lanes 10–18). Significantly,
as was the case for ATP hydrolysis, purified TIP49b
hexamers were found to be unable to unwind DNA
with a 30-nucleotide 3¢ extension (Fig. 3A, right,
compare lanes 20 and 22) or a 5¢ extension (data
not shown). Comparison of the unwinding time
courses obtained with TIP49b and Rvb2p (Fig. 3B)
demonstrates that they display similar apparent first-
order kinetics of unwinding under our experimental
AB
C
E
D
Fig. 2. ATPase and DNA binding activities of TIP49b and Rvb2p. (A) Nucleic acid requirement for ATP hydrolysis. TIP49b was incubated with
the nucleic acids shown (10 ngÆlL

)1
) in the presence of radiolabeled ATP (200 l M final) for 45 min at 37 °C before analysis by TLC (lanes 1–
5). The small accumulation of ADP above background detected in the presence of the protein alone indicates intrinsic ATPase activity (lane
2). (B) ATP hydrolysis by TIP49b monomers and hexamers in the presence of ssM13 DNA using purified TIP49b monomers (filled circles) or
hexamers (open circles). The inset shows the corresponding TLC for monomers (mono) and hexamers (hex) as a function of time (t). (C)
ATP hydrolysis as a function of ssDNA length: ssM13 (filled circles), ss115 (open circles), ss80 (filled triangles), ss50 (open triangles), ss21
(filled squares) or in the absence of ssDNA (open squares). Reaction mixtures contained a fixed concentration of protein (1 l
M) and 200 lM
ATP. (D) Length-dependent ssDNA binding by TIP49b. Binding reactions were performed with 21, 85 or 115 mer oligonucleotides (0.1 nM)
and increasing concentrations of TIP49b monomers. Binding dependence on ssDNA length follows that seen for ATP hydrolysis. (E) Compar-
ison between ssDNA and duplex DNA binding by TIP49b. Major and minor complexes are indicated by black and grey arrowheads, respec-
tively. The probable nature of the different complexes thus detected by electrophoresis on native 5% acrylamide gels is discussed in the
main text. Note that ds115 shows a higher mobility than ss115 on these gels. The use of 8% acrylamide gels restores the expected relative
mobilities of single-stranded and duplex DNA.
DNA unwinding by TIP49b C. Papin et al.
2708 FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS
conditions (k
app
= 0.24 ± 0.02 min
)1
for Rvb2p and
0.16 ± 0.02 min
)1
for TIP49b with 1 nm DNA sub-
strate, 1 lm protein, 1 mm ATP and 10 nm reanneal-
ing trap). We also measured unwinding activity as a
function of protein concentration, obtaining identical
curves, with a midpoint at 80.6 ± 3.6 nm for TIP49b
and 85.3 ± 4.7 nm for Rvb2p (Fig. 3C). It is impor-
tant to note that this similarity in terms of DNA

unwinding parallels the ATPase and DNA-binding
activities of both proteins, highlighting the remarkably
strong conservation of these three activities.
ssDNA-tail length requirement for DNA
unwinding by TIP49b and Rvb2p
The results reported above demonstrate that the
DNA-binding and ATPase activities of TIP49b are
sensitive to ssDNA length. This property might in turn
affect the efficiency of DNA unwinding. To test this,
we next constructed a set of 3¢-ss ⁄ ds junctions contain-
ing a 21 bp duplex region and 3¢ single-stranded exten-
sions of 0, 4, 19, 30, 39, 59 or 94 nucleotides
(Fig. 4A), and measured DNA unwinding activity
under our standard experimental conditions. The
results obtained in these experiments (Fig. 4B) reveal
that the efficiency of DNA unwinding by TIP49b and
Rvb2p depends on the length of the 3¢ single-stranded
tail. A sharp transition is observed between 19 and
39 nucleotides, with a midpoint at approximately
30 nucleotides of 3¢ single-stranded DNA (26 ± 2
nucleotides and 32 ± 4 nucleotides for TIP49b and
Rvb2p, respectively, as estimated by the sigmoid fit of
the data). Hence, short 3¢-protruding single-stranded
tails are not sufficient to trigger unwinding. However,
increasing the length of 3¢-protruding tails results in a
sharp activation above a threshold length of approxi-
A
BC
Fig. 3. DNA unwinding by TIP49b is preferentially exerted on 3¢-to5¢-ss ⁄ ds junctions. (A) Unwinding by 1 lM TIP49b (left, lanes 1–9) or
Rvb2p (center, lanes 10–18) was tested on DNA (1 n

M) in the absence of co-factors (lane 4) or in the presence of 1 mM AMP (lane 5), ADP
(lane 6), ATP (lane 7), AMP-PNP (lane 8) or ATP-c S (lane 9) for 30 min at 37 °C. Asterisks denote the 5¢ [P
32
]-label. 5¢-to3¢ and 3¢-to5¢ sub-
strates used to assay unwinding activities are shown to the left of the gels. The right panel (lanes 19–22) shows a comparison of 3¢-to5¢
DNA unwinding by purified TIP49b monomers (lanes 19–20) or hexamers (lanes 21–22). Lanes 19 and 21 are no-ATP controls. (B) Time
course of 3¢-to5¢ unwinding reactions performed with TIP49b (open and filled triangles show data from two independent experiments) or
Rvb2p (open circles). The lines are the best fit to a single exponential with an apparent first-order rate constant of 0.24 ± 0.02 min
)1
for
Rvb2p and 0.16 ± 0.02 min
)1
for TIP49b (deviations are from the fit). (C) Unwinding measurements performed at varying TIP49b (triangles)
or Rvb2p (open circles). Insets in (B) and (C) show representative experiments used for quantifications.
C. Papin et al. DNA unwinding by TIP49b
FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS 2709
mately 30 nucleotides. Additionally, we note that both
proteins were unable to unwind either blunt-ended
short DNA substrates, such as three- and four-way
junctions or nick-containing three-way junctions (data
not shown), consistent with the results obtained in
previously studies [16].
Duplex length affects the efficiency of DNA
unwinding by TIP49b
Although circular duplex DNA does not stimulate the
ATPase activity of TIP49b (Fig. 2A), we show next
that the protein is capable of binding to linear duplex
oligonucleotides. This property led us to test possible
effects of duplex DNA length on the unwinding effi-
ciency of TIP49b. We next constructed a set of 3¢-ss ⁄ ds

junctions containing a 30-nucleotide 3¢-tail and
progressively longer duplex regions of 21, 45, 55, 65
and 85 bp (ss30ds21, ss30ds45, ss30ds55, ss30ds65 and
ss30ds85; Fig. 4C). Reactions were performed with
1nm of DNA substrates, 100 nm TIP49b and 1 mm
ATP. Increasing the duplex length decreased the total
extent of unwinding and the overall reaction rate, with
a sharp drop between 45 and 65 bp (Fig. 4D), suggest-
ing that DNA unwinding occurs as a result of a short-
range activity of the protein on duplex DNA.
The observed drop in the efficiency of DNA
unwinding could be a result of reannealing of DNA
strands during or after the unwinding reaction.
Accordingly, the final amplitude of the reaction would
be underestimated. Indeed, the annealing rates of 21-
or 25-nucleotide oligonucleotides and ss115 at 37 °C
are in the range of 0.25 ± 0.03 to 0.41 ± 0.02 min
)1
,
respectively, at a nanomolar DNA concentration (data
AB
C
E
D
Fig. 4. DNA length requirement for DNA
unwinding by TIP49b. (A) Schematic presen-
tation of the DNA substrates containing 3¢
ssDNA tails of different lengths used in the
present study. Oligonucleotides used con-
tain a 21 bp duplex region and a 3¢ single-

stranded extension of 0, 4, 19, 30, 39, 59 or
94 nucleotides. Asterisks denote the 5¢
[P
32
]-label. (B) The DNA unwinding activity
of TIP49b or Rvb2p depends on the length
of the 3¢ single-stranded tail. Reactions
were performed for 30 min at 37 °C. Solid
circles: TIP49b; open circles: Rvb2p. Data
obtained from two independent experiments
for each protein were fitted with a sigmoid
curve. The inset shows representative bind-
ing experiments used for quantifications.
(C) 3¢-to5¢-ss ⁄ ds junctions containing a
30-nucleotide ssDNA tail and duplex regions
of different lengths. Oligonucleotides a–d
were used as traps, as described in the
main text, and are complementary to the
unlabeled top strand. (D) Time courses of
DNA unwinding of the substrates containing
duplex regions of different lengths. Filled
circles, ss94ds21; open circles, ss30ds21;
filled triangles, ss30ds45; open triangles,
ss30ds55; filled diamonds, ss30ds65; open
diamonds, ss30ds85. (E) Unwinding ampli-
tude versus duplex length in the absence of
in the presence of reannealing traps whose
permutations correspond in each case to
the extent of each duplex region tested
(filled circles and open circles, respectively).

DNA unwinding by TIP49b C. Papin et al.
2710 FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS
not shown), or approximately two- to three-fold faster
than the overall unwinding rate of TIP49b. To rule
out the possibility that the effects of duplex length we
observed are a result of reannealing occurring faster
than unwinding, yielding a drop in the overall mea-
sured unwinding efficiency, we repeated these experi-
ments in the presence of reannealing traps consisting
of short 20-nucleotide oligonucleotides complementary
to the unlabeled top strand (10 nm; Fig. 4E), with this
length being chosen because it does not support bind-
ing by TIP49b (Fig. 2D). The traps (a–d; Fig. 4C, bot-
tom) hybridize to the different duplex regions tested
and were added alone or in combination to match
each duplex. Because the addition of these permuta-
tions of the reannealing traps did not change the final
amplitude of DNA unwinding by TIP49b (Fig. 4E,
compare filled and open circles), we conclude that
reannealing of DNA during the reaction is not respon-
sible for the sharp decrease in the total extent of
unwinding seen for duplexes of 65 and 85 bp. As dis-
cussed below, these results suggest that the unwinding
properties of TIP49b documented in the present
study differ from those of processive hexameric-ring
helicases.
Discussion
We have studied the biochemical properties of mam-
malian TIP49b and yeast Rvb2p purified as monomers.
We found that the ATPase activity of TIP49b depends

on ssDNA in a length-dependent manner and can be
correlated with its ssDNA-binding properties. ATP
hydrolysis as a result of a contaminant can be ruled
out based on routine MS analysis of purified proteins
and a lack of other detectable ATP-binding activities
(Fig. 1D). In addition, the hexameric fraction of
TIP49b did not show detectable ATPase activity, nor
did it support DNA unwinding in our experimental
system. The availability of TIP49b ATPase mutants
would serve as a useful control as well as a powerful
tool to dissect the mechanisms that account for DNA
binding and unwinding. However, we and others have
failed to generate such mutants, and such a puzzling
failure has been discussed in a recent review [1]. Para-
doxically, in principle, the presence in TIP49b of pre-
sumptive essential motifs, such as Walker A and B
boxes, R-finger, and Sensor 1 and 2 motifs, should
allow for rational mutagenesis, although all efforts
have remained unsuccessful. We speculate that the
controlled and ssDNA-dependent ATPase activity of
TIP49b is unusually sensitive to protein conforma-
tional changes that might be induced by ATP binding.
In this case, attempts to mutate ATP binding pockets
might lead to more active rather than inactive
mutants.
We demonstrate that both TIP49b and yeast Rvb2p
possess a slow ATP-dependent strand separation activ-
ity, which is exerted on 3¢-ss ⁄ ds junctions containing a
protruding single-stranded tail ‡ 30 nucleotides. The
results obtained suggest that, on these substrates,

DNA unwinding by TIP49b occurs as a result of pro-
tein action over short distances along duplex DNA,
whose length affects the efficiency of strand separation.
Given a 3¢-protruding tail of fixed length, the extent of
strand separation is inversely correlated with the length
of duplex DNA lying beyond the junction (Fig. 4D,
E), indicating a mechanism of action strictly conserved
between mammalian and yeast TIP49b proteins, but
differing from that of processive hexameric-ring heli-
cases. Because TIP49b also binds duplex DNA, a sim-
ple explanation would be that distinct interactions of
monomers bound to ssDNA or adjacent duplex
DNA ‡ 45 bp intrinsically limit the extent of the
unwinding reaction. The molecular basis for such a
limitation remains unknown at present, although it
could implicate ATP-induced conformational changes
undergone by TIP49b at ss- ⁄ dsDNA junctions. Further
analysis of the nature of the complexes formed by
TIP49b with ssDNA or dsDNA will be required to
address this question in more detail.
The strict 3¢-to5¢ directionality of DNA unwinding
by TIP49b and Rvb2p documented in the present
study on well defined substrates is consistent with that
of the TIP60 and Ino80 chromatin-remodeling com-
plexes containing TIP49a and TIP49b. However, it dif-
fers from the initial report on the properties of purified
rat TIP49b on much longer ssDNA substrates [13].
Using similar ssM13-helicase substrates and high pro-
tein concentrations, we also detected both 3¢-to5¢ and
5¢-to3¢ unwinding by TIP49b and Rvb2p (data not

shown). Although it is difficult to compare these out-
comes given the significant differences in experimental
conditions, our observation that short DNA substrates
and low protein concentrations support a 3¢-to5¢
polarity of unwinding by TIP49b and Rvb2p could
indicate that the ratio of ss- to dsDNA in these differ-
ent substrates influences the polarity of DNA strand
separation. We also note that a previous study of
Rvb2p did not reveal a detectable unwinding activity
using 3¢-to5¢ or 5¢-to3¢-ss ⁄ ds junctions (composed of
a 15-nucleotide ss-tail and 28 bp duplex), even at high
(3 lm) protein concentrations [15]. However, this
apparent discrepancy is consistent with our results
showing the strong dependence of unwinding on the
length of the ssDNA tail, suggesting that 15-nucleotide
tails are not long enough to support efficient DNA
C. Papin et al. DNA unwinding by TIP49b
FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS 2711
unwinding by TIP49b and Rvb2p (Fig. 4B). Finally,
we note the possibility that TIP49b ⁄ TIP49a hetero-
hexamers might exhibit characteristic properties. This
has been previously investigated [15], but using protein
concentrations in the range 5–40 lm. The use of such
concentrations for TIP49b alone leads, in our hands,
to protein aggregation and we are currently attempting
to develop new approaches conducive to controlled
hexamerization, which might also be extended to the
study of TIP49a ⁄ TIP49b interactions.
Because the protein purification protocol used in the
present study yields both monomeric and hexameric

fractions of TIP49b (Fig. 1), we also analyzed hexa-
mers side-by-side with monomers. TIP49b hexamers
showed some residual DNA binding activity in
gel-shift assays but supported neither ATP hydrolysis
(Fig. 2B), nor DNA unwinding (Fig. 3A). Hence,
hexameric TIP49b assemblies appear to be inactive.
Because hexamers would ultimately need to be con-
verted back into an active form of the protein, these
findings, if biologically relevant, suggest that yet
unknown protein cofactor(s) might be required to
recycle TIP49b hexamers in cells.
Materials and methods
Protein purification
A pET3a vector containing the coding sequence of human
TIP49b was a kind gift from V. Ogryzko (IGR, Villejuif,
France) [9]. The protein was expressed in the BL21(DE3)
pLysS E. coli strain. A 2 L culture was grown in LB med-
ium at 37 °C until D
600
of 0.5 was reached before induction
with 1 mm isopropyl thio-b-d-glactoside for 6 h at 25 °C.
Cells were lysed in 100 mL of a buffer containing 20 mm
Tris–HCl (pH 8.0), 500 mm NaCl, 10% glycerol, 1 mm
dithiothreitol and 10 mm imidazole for 30 min on ice in the
presence of lysozyme at 1 mgÆmL
)1
and protease inhibitor
cocktail tablets (Roche Diagnostics, Basel, Switzerland) and
sonicated on ice for 3 · 10 s. The clarified supernatant was
applied to a Ni

2+
-sepharose column (HisTrapÔ FF, 5 mL;
GE Healthcare, Milwaukee, WI, USA) equilibrated with
the same buffer, then washed in the presence of 10 mm
imidazole and subjected to two step elutions with 100 and
500 mm imidazole using a buffer containing 20 mm Tris–
HCl (pH 8.0), 100 mm KCl, 10% glycerol and 1 mm dith-
iothreitol. Two TIP49b-containing fractions were detected.
Fraction ‘low imidazole’ was eluted from the column dur-
ing the 100 mm imidazole step, whereas the rest of the pro-
tein eluted at 500 mm (‘high imidazole’). Gel filtration on a
HiLoad 16 ⁄ 60 Superdex S-200 PG column (GE Healthcare)
followed by SDS ⁄ PAGE revealed that the ‘low imidazole’
fraction contained monomers used throughout the study
after gel filtration (fraction 2), hexamers (fraction 1) and
high molecular-weight aggregates. Where indicated, TIP49b
hexamers were from fraction 1. TIP49b and Rvb2p prepa-
rations were routinely controlled by LC-MS ⁄ MS for the
absence of contamination by bacterial ATPases and heli-
cases. In all cases, pooled purified monomer and hexamer
fractions were quantified using the Bradford assay and
aliquots were used directly without additional concentra-
tion.
The pET-9aSN1 vector (a kind gift from S. Cheruel,
IBBMC, University Paris-Sud, Orsay, France) containing
the coding sequence of the yeast Rvb2 protein was used to
transform E. coli BL21(DE3) STAR. Transformed cells
were grown in 2TY medium at 37 °C until D
600
of 0.8 was

reached. Expression of C-terminal His-tagged Rvb2p was
induced with 0.5 mm isopropyl thio-b-d-glactoside for 3 h.
Cells were lysed in 50 mm Tris–HCl (pH 7.5), 300 mm
NaCl and 5 mm b-mercaptoethanol. After sonication and
centrifugation for 30 min at 25 000 g, the clarified lysate
was applied to a Fast-Flow Ni-NTA agarose column
(Qiagen, Valencia, CA, USA). Rvb2p was eluted with a
10–300 mm imidazole gradient in lysis buffer. Pooled peak
fractions were diluted ten-fold in buffer containing 25 mm
Bis-Tris propane (pH 6.5), 5 mm b-mercaptoethanol and
loaded onto a MonoQ 5 ⁄ 50 GL column (GE Healthcare).
Elution was performed with a 0–1 m NaCl gradient. Frac-
tions containing pure Rvb2p were collected and dialyzed
against 20 mm Tris–HCl (pH 8.0), 100 mm KCl, 10% glyc-
erol and 1 mm dithithreitol. Rvb2p preparations were
found to be a mixture of dimers and monomers, as judged
by gel filtration.
TIP49b and aFLAG antibodies were purchased from BD
Biosciences (Franklin Lakes, NJ, USA, USA) and from
Sigma (St Louis, MO, USA), respectively. RecA protein
was purchased from New England Biolabs (New England
Biolabs, Beverly, MA, USA).
DNA substrates
The DNA substrates for gel-shift experiments and helicase
assays were prepared by annealing of equimolar amounts
of the corresponding synthetic oligonucleotides in a buffer
containing 10 mm Tris–HCl (pH 7.5), 1 mm EDTA and
100 mm NaCl and analyzed by native gel electrophoresis.
The specificity of DNA unwinding is defined here by the
protruding ss-tail. The oligonucleotides used are shown in

Table S1.
ATPase assays
The ATPase activity of the proteins (1 lm) was measured
in a 5 lL reaction volume containing 25 mm Hepes-KOH
(pH 8.0), 2.5 mm Mg(CH
3
COO)
2
, 100 mm KCl, 0.2 mm
dithiothreitol, 100 lgÆmL
)1
BSA (Sigma) and 0.6 lCiÆlL
)1
[a-P
32
] ATP, and unlabeled ATP up to 2 mm. This reaction
buffer was used throughout the study. The reaction
DNA unwinding by TIP49b C. Papin et al.
2712 FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS
mixtures were incubated at 37 °C for 0, 2, 5, 10 and
30 min, stopped on ice and analyzed by TLC on PEI-Cellu-
lose plates (Merck, Darmstadt, Germany) using 0.75 m
KH
2
PO
4
as a migration buffer; plates were then dried and
quantified.
DNA binding
TIP49b or Rvb2p recombinant proteins at 0–1 lm concen-

trations were incubated in a 10 lL reaction volume con-
taining 25 mm Hepes-KOH (pH 8.0), 2.5 m m
Mg(CH
3
COO)
2
, 100 mm KCl, 0.2 mm dithiothreitol and
100 lgÆmL
)1
BSA (Sigma). DNA substrates at the indicated
concentrations were incubated for 45 min and electrophore-
sed on native 8% polyacrylamide gels (19 : 1) using
1 · TBE as a running buffer. Gels were dried and quanti-
fied on a Fuji BAS 3000 phosphorimager (Fuji Life
Sciences, Tokyo, Japan).
Helicase assays
5¢-to3¢ or 3¢-to5¢ helicase substrate (1 nm) was preincu-
bated with 0.1 or 1 lm TIP49b or Rvb2p as indicated for
15 min on ice. The reaction mixtures were complemented
or not with 1 mm nucleotide cofactor, as indicated, and
incubated at 37 °C. A ten-fold excess of a trap oligonucleo-
tide (complementary to the unwound radio-labeled probe)
was added to the reaction mixture to prevent reannealing.
The reactions were stopped by the addition of 2 lLofa
solution containing 1 mgÆmL
)1
proteinase K, 1.25% SDS,
10 mm Tris–HCl, 0.06% bromophenol blue, 0.06% xylene
cyanol and 30% glycerol in the presence of 100-fold excess
of the trap oligonucleotide. The samples were analyzed by

native electrophoresis on 8% polyacrylamide gels using
1 · TBE as running buffer. Gels were dried and quantified
on a Fuji BAS 3000 phosphorimager.
Electron microscopy
TIP49b or Rvb2p (2 lm) were spread on carbon-coated
copper grids (100 or 400 mesh). After 30–60 s, the drops
were blotted dry and the grids were stained with 1% uranyl
acetate for 1 min, blotted again, and washed or not once in
water for 30 s. Grids were dried and examined with a trans-
mission electron microscope (ME Hitachi 200 kV; Hitachi,
Tokyo, Japan).
Acknowledgements
We thank Simon Lebaron for RNA samples; Ross
Tamaino for mass spectroscopy; Ste
´
phanie Balor and
Nathalie Laviolette for technical help; Patrick Schultz
for help with electron microscopy; Dave Lane for
comments on the manuscript; and Martine Obadia for
helpful discussions. This work was supported by
grants from the Agence Nationale pour la Recherche
(ANR grant ‘DNAMOTORS’ No. 143704) and the
Association pour la Recherche sur le Cancer (ARC)
to M.G. and E.K., Universite
´
Paul Sabatier and the
Centre National de la Recherche Scientifique (CNRS).
K.E. was supported by a grant from the Agence
Nationale pour la Recherche (ANR-05-JCJC
No. 015101) to S.M. C.P. and A.K. were recipients of

PhD fellowships from the French Ministry for
Research. C.P. received additional fellowship support
from the ARC.
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Supporting information
The following supplementary material is available:
Table S1. Oligonucleotides used in this study.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
DNA unwinding by TIP49b C. Papin et al.
2714 FEBS Journal 277 (2010) 2705–2714 ª 2010 The Authors Journal compilation ª 2010 FEBS