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

In the present study, we show that both human TIP49b and its yeast orthologue, Rvb2p, cooperatively bind single-stranded DNA as monomers.. Although TIP49b black arrowheads showed a lower

Christophe Papin1,2,*,, Odile Humbert1,2,*, Anna Kalashnikova1,2, Kelvin Eckert3, Solange Morera3, Emmanuel Ka¨s1,2and Mikhail Grigoriev1,2

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] Bedur-ing 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: grigor@ibcg.biotoul.fr;

kas@ibcg.biotoul.fr

*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¢- to 5¢ 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 ) and TIP49b (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.

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 transacfunc-tions 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 funcinterac-tions 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⁄ His6- and C-terminal His6-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

Monomer Hexamer

TIP49b:

0 20

100 nm 100 nm

40 60 80 100 mL

20

A

B

15

10

5

0

2000 440 158 67 43 kDa

TIP49b RecA – + + +

1 2 3

ssM13

kDa

1 2

55 36 31 21

66 97

Western blotting Silver 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 [cP32]-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.

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-P32]-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-P32]-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

Reactions were allowed to proceed at 37C 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

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 l M

ATP (D) Length-dependent ssDNA binding by TIP49b Binding reactions were performed with 21, 85 or 115 mer oligonucleotides (0.1 n M ) 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.

conditions (kapp= 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 unwindreanneal-ing 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¢ scontain-ingle-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

Fig 3 DNA unwinding by TIP49b is preferentially exerted on 3¢- to 5¢-ss ⁄ ds junctions (A) Unwinding by 1 l M 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 m M AMP (lane 5), ADP (lane 6), ATP (lane 7), AMP-PNP (lane 8) or ATP-cS (lane 9) for 30 min at 37 C Asterisks denote the 5¢ [P 32 ]-label 5¢- to 3¢ and 3¢- to 5¢ 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¢- to 5¢ 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¢- to 5¢ 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)1for Rvb2p and 0.16 ± 0.02 min)1for 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.

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

1 nm 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 unwindsuggest-ing 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 37C are in the range of 0.25 ± 0.03 to 0.41 ± 0.02 min)1, respectively, at a nanomolar DNA concentration (data

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¢ [P32]-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¢- to 5¢-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).

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¢- to 5¢ 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¢- to 5¢ and 5¢- to 3¢ 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¢- to 5¢ 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¢- to 5¢ or 5¢- to 3¢-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

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 monohexa-mers TIP49b hexahexa-mers

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 37C until D600of 0.5 was reached before induction

with 1 mm isopropyl thio-b-d-glactoside for 6 h at 25C

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 Ni2+-sepharose column (HisTrap 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

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

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 37C until D600of 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

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

dithiothreitol, 100 lgÆmL)1 BSA (Sigma) and 0.6 lCiÆlL)1 [a-P32] ATP, and unlabeled ATP up to 2 mm This reaction buffer was used throughout the study The reaction

mixtures were incubated at 37C 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

KH2PO4as 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

100 lgÆmL)1BSA (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¢- to 3¢ or 3¢- to 5¢ 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 37C 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 lL of a

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

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