Báo cáo khoa học: The Cockayne syndrome group B protein is a functional dimer docx

Recently, the structure of the central ATPase domain of zebrafish Rad54 revealed that the conserved core of this SWI2⁄ SNF2 protein is similar to SF2 heli-cases [9].. The integrity of the

Mette Christiansen1, Tina Thorslund1, Bjarne Jochimsen2, Vilhelm A Bohr3and Tinna Stevnsner1

1 Danish Centre for Molecular Gerontology, Department of Molecular Biology, University of Aarhus, Denmark

2 Department of Molecular Biology, University of Aarhus, Denmark

3 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

Cockayne syndrome (CS) is a segmental premature

aging syndrome with complex symptoms, including

developmental abnormalities, neurological dysfunction,

and short average lifespan Cellular characteristics

include hypersensitivity to UV light, and failure of

RNA synthesis to recover to normal rates following

UV irradiation Two genes have been shown to be

involved: CSA and CSB [1] The CSB gene encodes a

protein with a predicted molecular mass of 168 kDa

The CS group B (CSB) protein contains an acidic

domain, a glycine-rich region, and two putative

nuc-lear localization signal (NLS) sequences [2] In

addi-tion, CSB is a member of the SWI2⁄ SNF2-family of

DNA-dependent ATPases that contain seven

charac-teristic motifs which are also present in DNA and

RNA helicases [3] Helicase activity has not been dem-onstrated for any members of the SWI2⁄ SNF2-family, which is part of Superfamily 2 (SF2), but in general they have the ability to destabilize protein–DNA inter-actions [4] The CSB protein displays DNA-dependent ATPase activity and CSB is able to remodel chromatin

in vitro[5–8]

Recently, the structure of the central ATPase domain of zebrafish Rad54 revealed that the conserved core of this SWI2⁄ SNF2 protein is similar to SF2 heli-cases [9] This indicates that SWI2⁄ SNF2 proteins translocate on DNA with a mechanism similar to heli-cases The integrity of the SWI2⁄ SNF2 ATPase domain is critical for most functions of CSB in vitro and in vivo Mutations in motif Ia, II, V, and VI either

Keywords

Cockayne syndrome group B protein;

DNA-dependent ATPase; homodimer;

SWI2 ⁄ SNF2; transcription coupled repair

Correspondence

T Stevnsner, Danish Centre for Molecular

Gerontology, Department of Molecular

Biology, University of Aarhus, Build 130,

DK-8000 Aarhus C, Denmark

Tel: +45 89422657

Fax: +45 89422650

E-mail: tvs@mb.au.dk

(Received 13 May 2005, revised 1 July

2005, accepted 4 July 2005)

doi:10.1111/j.1742-4658.2005.04844.x

Cockayne syndrome (CS) is a rare inherited human genetic disorder char-acterized by developmental abnormalities, UV sensitivity, and premature aging The CS group B (CSB) protein belongs to the SNF2-family of DNA-dependent ATPases and is implicated in transcription elongation, transcription coupled repair, and base excision repair It is a DNA stimula-ted ATPase and remodels chromatin in vitro We demonstrate for the first time that full-length CSB positively cooperates in ATP hydrolysis as a function of protein concentration We have investigated the quaternary structure of CSB using a combination of protein–protein complex trapping experiments and gel filtration, and found that CSB forms a dimer in solu-tion Chromatography studies revealed that enzymatically active CSB has

an apparent molecular mass of approximately 360 kDa, consistent with dimerization of CSB Importantly, in vivo protein cross-linking showed the presence of the CSB dimer in the nucleus of HeLa cells We further show that dimerization occurs through the central ATPase domain of the pro-tein These results have implications for the mechanism of action of CSB, and suggest that other SNF2-family members might also function as dimers

Abbreviations

CS, Cockayne syndrome; CSB, CS group B; HA, hemaglutinin antigen; HIS, His6; SF1, superfamily 1; NLS, nuclear localization signal; NTB, nucleotide binding fold; SF2, superfamily 2.

abolish or drastically reduce the ATPase activity of

CSB [7,10] CSB cDNA with point mutations in motifs

Ia, II, III, V, and VI, as opposed to wt CSB cDNA,

do not complement the deficiencies of the SV40

trans-formed CS-B cell line, CS1AN.S3.G2 [11–13] In

con-trast, both a deletion of the entire acidic region of 39

amino acids and a point mutation in a putative

nucleo-tide binding (NTB) motif do not interfere with the

ability of CSB to complement CSB-deficient cells

[12,14,15]

The majority of bacterial and viral DNA helicases

appear to act as oligomers, usually dimers or hexamers

[16] Consequently, it is tempting to speculate that

members of the SWI2⁄ SNF2 of DNA-dependent

ATP-ases might also function as multimers Recent results

indicate that the Swi2p ATPase subunit is present in a

single copy in the yeast SWI⁄ SNF chromatin

remodel-ing complex [17] In contrast, yeast Rad54, which is

involved in recombination, seems to be a monomer in

solution and a dimer⁄ oligomer on DNA [18] Insight

into the quaternary structure of CSB will advance the

understanding of the mechanism by which the

DNA-dependent ATPases, in general, and CSB, in particular,

functions Furthermore, oligomerization status is

important to evaluate the stoichiometry of different

biochemical analyses The three-dimensional structure

of CSB has not yet been elucidated, and we report here

a characterization of CSB protein structure We find

that the CSB protein forms a dimer in vitro and in vivo,

and that this homodimerization is essential for ATP

hydrolysis of CSB Moreover, we demonstrate that the

ATPase domain is involved in the dimerization

Results

CSB ATP hydrolysis exhibits

non-Michaelis-Menten kinetics

In general, DNA helicases often function as oligomers

[16] Because CSB belongs to the superfamily 2 of

heli-cases, it is of importance to investigate whether CSB

may also function as an oligomer Initially, the dose–

response curve for ATP hydrolysis previously reported

[10] was reexamined in more detail using low levels of

CSB protein Figure 1A shows that product formation

is not linear with increasing concentrations of CSB

protein, suggesting positive cooperativity in ATP

hydrolysis by CSB Thus, these results suggest that the

CSB protein, under the experimental conditions used,

functions as a multimer Furthermore, the Hill

coeffi-cient of 2.1, which is the maximum slope from the Hill

plot (Fig 1B), clearly indicates positive cooperativity,

suggesting that CSB acts as a dimer

CSB displays homodimerization in solution

in vitro

To test the dimerization in further detail, we per-formed cross-linking in solution to trap the CSB homodimer This is a sensitive and widely used method for in vitro analysis of protein–protein interactions [19,20] We found that recombinant purified CSB at low concentration in solution could be cross-linked with glutaraldehyde The cross-linked species were identified with silver stain, and the apparent molecular mass of 330 kDa was determined from the migration

0.4

y = 2.1x + 7.9

-1.0 -0.5 0.0 0.5 1.0 1.5

Log[ATP]

-0.1 0.0 0.1 0.2 0.3

0.5 0.6

CSB (nM)

A

B

Fig 1 Effect of increasing amounts of CSB on its ATPase activity (A) [32P]ATP[cP] hydrolysis rate after incubation with 0–6 nM recom-binant CSB, 50 lM cold ATP and 150 ng plasmid DNA for 1 h at

30
C Error bars represent standard deviations of three independ-ent experimindepend-ents (B) ATP hydrolysis rate was determined for 6 nM CSB incubated with increasing amounts of ATP Graph shows a Hill plot of a representative experiment.

of the molecular mass standards Given a predicted

subunit molecular mass of 168 kDa, this corresponds

well with a homodimer of CSB (Fig 2A)

Further-more, cross-linking also resulted in aggregation in the

slot Interestingly, the presence of ATP, ATP[cS],

co-factor DNA, or dephosphorylation of CSB with

protein phosphatase 1 did not have any effect on the

extent of dimerization in solution (Fig 2B and not shown)

Gel filtration reveals enzymatic activity of the CSB dimer

In order to characterize the quaternary structure of the CSB protein, we carried out gel filtration The CSB protein eluted as a peak around fraction 24 from a Superdex 200 column (Fig 3) as determined by DNA-dependent ATPase activity measured in the different fractions On the basis of the elution of the molecular mass markers, this peak corresponds to a molecular mass of approximately 360 kDa Given a predicted subunit molecular mass of 168 kDa, this indicates that CSB is a dimeric protein DNA was not present in these fractions since the ATPase activity was only detectable after the addition of pUC19 DNA This indicates that dimerization is not mediated by DNA Importantly, only residual ATPase activity was observed at the monomer size (fraction 27), while sil-ver staining of SDS⁄ PAGE clearly showed elution of CSB at this position (Fig 3, compare fractions 25 and 27) This suggests that CSB is only active as an ATPase when it is a dimer Also, we did not detect a peak in DNA-dependent ATPase activity at fractions earlier than the ferritin marker (450 kDa), suggesting that CSB does not exist as higher order oligomers in solution

CSB exhibits homodimerization in vivo Next, we tested whether the dimerization observed in solution in vitro and its stimulating effects on CSB enzy-matic activity are biologically relevant HeLa cells were exposed to a range of formaldehyde concentrations in

an attempt to covalently cross-link endogenous CSB Nuclear extracts were prepared and proteins were ana-lyzed by western blotting using CSB-specific antibody Besides the CSB monomer, only a single CSB complex was detected in western blot from the nuclear extract after treatment of cells with 10 mm formaldehyde This CSB complex migrated to the position of a CSB dimer

in SDS⁄ PAGE (Fig 4A) Furthermore, both bands are specific to CSB as both the monomeric and the dimeric bands were absent in extracts from CS1AN.S3.G2 cells which lack full-length CSB (Fig 4A) Next, we analyzed whether the fraction of CSB dimer compared to mono-mer increased after UV irradiation or transcription inhi-bition by a-amanitin, but we did not see any effect (Fig 4B) It remains to be determined whether other factors, such as oxidative damage, affect the extent of CSB dimerization in vivo

Fig 2 Stabilization of the CSB dimer by in vitro protein-protein

cross-linking with glutaraldehyde (A) CSB (60 nM) was incubated

with 0.001% (v ⁄ v) glutaraldehyde in solution for 0, 10, 20 or

40 min, and CSB was detected by 3–8% (w ⁄ v) Tris ⁄ acetate

SDS ⁄ PAGE and silver stain (B) CSB was incubated with 0.001%

(v ⁄ v) glutaraldehyde in the presence or absence of 50 lM ATP or

ATPcS as indicated CSB was detected by 3–8% (w ⁄ v) Tris ⁄ acetate

SDS ⁄ PAGE and western blot with CSB specific antibody *CSB

monomer; **CSB dimer The size (in kDa) of a protein marker is

indicated.

CSB forms a homodimer through the

DNA-dependent ATPase domain

To map which part of CSB mediates homodimerization,

we carried out interaction studies of recombinant

wild-type CSB [N-terminal hemaglutinin antigen (HA) and

C-terminal His6 (HIS) tagged] with CSB fragments

(N-terminal S- and HIS- tags and C-terminal HIS- and

HSV tags) Five tagged fragments covering the entire

region of CSB; CSB(2–341), CSB(310–520), CSB(465–

1056), CSB(953–1204), and CSB(1187–1493) were used

(Fig 5A) The fragments were expressed in Escherichia

coli, purified, and mixed with purified wild-type CSB

In vitropull down experiments using S-protein-agarose

were performed and analyzed by western blot and use of

HA and HSV antibodies The result shown in Fig 5B

indicates that the protein homodimerizes through

inter-actions with the ATPase domain The CSB(465–1056)

fragment, which covers the SWI⁄ SNF-domain, interacts

tightly with the full-length CSB protein (Fig 5B, lane

3) Approximately 10% of input full-length CSB was

pulled down by the CSB(465–1056) fragment

Import-antly, purified wild-type CSB did not bind to

S-protein-agarose and there was little or no interaction with the

four other fragments (Fig 5B) The fragments were all

present in similar amounts in the pull-down experiment

as shown in the lower panel of Fig 5B

Discussion

In this report we present evidence that CSB forms a

dimer in vitro and in vivo Most bacterial and viral DNA

helicases appear to act as oligomers, usually dimers or hexamers, providing the helicase with multiple DNA binding sites [16] Recently, the Bloom’s syndrome heli-case was also identified as forming an oligomeric ring structure [21] This was the first example of oligomer formation of a helicase of human origin Multimeriza-tion has previously been reported for the Saccharomyces cerevisiaeSWI2⁄ SNF2 family member Rad54, and only

in the presence of DNA [18] A very recent paper des-cribes that the CSB protein wraps DNA around its sur-face and ATP hydrolysis leads to unwrapping Size analysis of scanning force microscopy pictures of DNA-bound CSB indicated a size of approximately 270 kDa, which lies between monomer and dimer size [22] Here,

we demonstrate for the first time that the purified recombinant CSB protein in fact displays biochemical characteristics that show that the protein functions as a dimer, and that CSB exists as a dimer in solution In addition, we show that endogenous CSB protein forms

a homodimer in vivo and that homodimerization occurs via the central ATPase domain of the CSB protein

Enzymatic evidence for dimerization Initially, a nonlinear dose–response curve indicated cooperativity of ATP hydrolysis and thus that CSB was acting as an oligomer The Hill coefficient of 2.1 suggested that at least two binding sites participate in the catalytic activity This is similar to results obtained for the ATPase activity of MJ0796, an ATP-binding cassette transporter, which forms homodimers in the presence of ATP [23] Trapping experiments with

0 2 4 6 8 10 12 14 16

fraction

CSB

Fig 3 Size-exclusion chromatography of

CSBATPase activity of fractions after elution

from Superdex 200 The elution positions of

the following markers are shown: ferritin

(450 kDa), glutamate dehydrogenase (GDH,

320 kDa), catalase (253 kDa) and lactate

dehydrogenase (LDH, 135 kDa) SDS ⁄ PAGE

(7%, w ⁄ v) and silver stain of Superdex

frac-tion 24–28 is shown in the lower panel, the

darker appearance of fraction 24 is due to

the coelution of marker protein (ferritin) in

this fraction.

glutaraldehyde of the CSB dimer showed that CSB

exists as a dimer in solution and indicated that the

dimer forms in the absence of DNA and ATP In

fur-ther support of CSB acting as a multimer, it has been

reported that structural mononucleosome alterations

needed a CSB to core particle ratio of about 4 : 1 [8]

Further, CSB was shown to be present in a large

molecular mass complex of > 700 kDa in gently

puri-fied HeLa whole cell extracts [24] The exact nature of

the complex was not determined, however, RNAPII

seemed to elute at the same size These results were

confirmed in a more recent report, which suggested

that GFP tagged CSB resides in a high molecular mass

complex (> 800 kDa) in living cells [25] These results

corroborate the existence of a CSB dimer, but also

suggest that the CSB dimer associates with other pro-teins to form a larger complex in vivo The inability to detect other protein complexes in the current study by formaldehyde cross-linking in vivo may indicate that such complexes cannot be cross-linked with formalde-hyde, or that only a small proportion of CSB protein

is part of other complexes

Dimerization is important for CSB ATPase activity

The quaternary structure of the CSB protein was further analyzed by gel filtration chromatography of recombinant purified CSB protein, and ATPase activity was monitored in parallel to assess where active CSB eluted These experiments showed that the enzymatic activity of the purified CSB protein elutes at the size of

a CSB dimer, and notably, only residual activity was found at the monomer size This is in contrast to results obtained for the Bloom’s syndrome helicase (BLM) oligomeric ring, where it was demonstrated that a minor peak of activity eluted at the monomer size [21]

We also show that endogenous CSB exists as a dimer in vivo in HeLa cells, thus supporting the signifi-cance of the in vitro observations of dimerization Only

a small fraction of the CSB protein was found to dimerize in vivo, and concurrently we found that the monomer only exhibited reduced ATPase activity This suggests that there might be an equilibrium between monomeric, ATPase inactive, and dimeric, ATPase active, forms of CSB, and raises the question of what role the enzymatic inactive monomer form might play inside a cell Previously, we have shown that a motif II CSB mutant deprived of ATPase activity retained the potential to partially complement the deficiency in incision at 8-oxoG [10,26] Thus, it seems likely that ATPase inactive forms of CSB may be important for its function in the repair of oxidative damage

Importantly, we find that homodimerization likely occurs via the central, conserved ATPase domain Interestingly, it has been reported that rad50, which is involved in double-strand break repair, dimerizes through interaction between the Walker A and Walker

B motifs in opposing subunits [27] These motifs are homologous to motif I and II, respectively, in CSB and thus supports the possibility of CSB dimerization through the ATPase domain

In the case of helicases, dimerization is of clear benefit for the processivity of the helicase reaction, such that alternating subunits can be engaged in unwinding the DNA duplex or tethering the enzyme to product single stranded DNA at the expense of ATP hydrolysis However, what role might dimerization

250

150

**

*

100

75

p89

250

150

**

*

p89

control UV α-amanitin control UV α-amanitin

100

75

A

B

Fig 4 In vivo cross-linking of the dimeric CSB complex with

for-maldehyde in HeLa cells Western analysis with the CSB specific

antibody of (A) nuclear extracts from HeLa and CS1AN cells

cross-linked with 0 or 10 mM formaldehyde, top panel shows analysis

with CSB specific antibody, while lower panel shows the same

western blot probed with p89 antibody and indicates equal loading.

(B) Nuclear extracts from control, UV-irradiated, or a-amanitin

trea-ted and formaldehyde (0 or 10 mM) cross-linked HeLa cells *CSB

monomer; **CSB dimer, size (in kDa) of a protein marker is

indica-ted, lower panel shows the same blot probed with p89 antibody.

have for a protein that does not act as a helicase but

as a chromatin remodeller? In this case it can be

specu-lated that the presence of multiple DNA and protein

binding sites due to dimerization of CSB in the same

manner increases the processivity of the enzyme, and

enables alternation in subunit interaction with DNA

and histones In addition, different subunits of the

CSB dimer may interact with distinct interaction

part-ners thus creating a link between processes such as

transcription and repair We speculate that the

dimeri-zation may play an important role in patients

expres-sing mutant forms of CSB with expres-single amino acid

substitutions [28] These mutations may affect the

dimerization and thus impair the activity of CSB This,

however, needs to be investigated further

Our in vitro experiments, using recombinant CSB protein, indicate that dimer formation involving the ATPase domain might be an allosteric effector for positive cooperativity Because we detected the CSB dimer in vivo in the presence of other CSB-interact-ing proteins, we propose that dimerization plays an important role in the regulation of its activity in the cell

Experimental procedures Recombinant proteins

Recombinant CSB wt protein containing an N-terminal hemaglutinin antigen (HA) epitope and a C-terminal HIS

116

34

CSB CSB CSB CSB CSB

IV

197

65

αHA

αHSV

2-341 310-520 465-1056 953-1204 1187-1493

S-protein agarose

1

A

B

2-341

310-520

465-1056

953-1204

1187-1493

Fig 5 The homodimerization of CSB depends on the DNA-dependent ATPase domain (A) Schematic representation of full-length CSB and CSB fragments used to map the homodimerization Full-length CSB contains an acidic domain (Ac), a glycine rich region (G), two nuclear localization signals (NLS), a putative nucleotide binding fold (NTB), and the seven conserved DNA-dependent ATPase motifs (I, IA and II to VI) The five CSB fragments cover amino acids 2–341, 310–520, 465–1056, 953–1204, and 1187–1493 of CSB, respectively (B) The CSB fragments were expressed in E coli and purified The CSB fragments were bound to S-protein agarose and subsequently incubated with wild-type CSB The beads were washed extensively and analyzed by SDS ⁄ PAGE and western Precipitated full length HSV CSB was visual-ized with HA antibody, while the tagged CSB fragments were visualvisual-ized by antibody Size (in kDa) of molecular mass markers is indicated.

tag was purified from insect cells as previously described

[10] The cloning, expression, and purification of CSB

frag-ments will be described elsewhere Briefly, the five CSB

fragments were amplified by PCR and cloned into the

pTriEx-4 Neo vector (Novagen, Madison, WI, USA) This

vector encodes N-terminal S- and HIS- tags and C-terminal

HIS- and HSV-tags The fragments were over expressed in

E coliand purified using Ni-NTA agarose (Qiagen,

Valen-cia, CA, USA)

CSB ATPase activity

The ATPase activity of CSB was determined as

previ-ously described [10] Standard reactions (10 lL) were

per-formed with 150 ng DNA cofactor, supercoiled (> 90%)

pUC19 plasmid, and 1 lCi [32P]ATP[cP] (3000 Ci

mmol)1, Hartmann Analytic, Braunschweig, Germany) in

buffer B (20 mm Tris⁄ HCl pH 7.5, 4 mm MgCl2, 50 lm

ATP, 40 lgÆmL)1 BSA, 1 mm dithiothreitol) Reactions

were incubated for 1 h at 30
C and stopped by the

addi-tion of 5 lL 0.5 m EDTA Samples (1 lL) were analyzed

on a polyethylenimine⁄ cellulose thin layer

chromatogra-phy plate developed in 0.75 m KH2PO4 Plates were

exposed on screen and ATP hydrolysis was analyzed

using a Molecular Imager For determination of the Hill

coefficient 6 nm of CSB protein was used, while the

amount of substrate was varied between 100 and 350 lm

Less than 20% of the ATP was hydrolyzed during the

incubations

Gel filtration

Sepharose CL 6B and Superdex 200 columns (50 mL,

Amersham Pharmacia, Piscataway, NJ, USA) were used

at 4
C with buffer A [25 mm Hepes–KOH pH 7, 0.01%

(v⁄ v) NP-40, 10% (v ⁄ v) glycerol, 1 mm

2-mercaptoetha-nol, 0.1 mm phenylmethylsulfonyl fluoride, 0.3 m KCl] as

elution buffer Samples of 100 lg homogeneous CSB

pro-tein (at an approximate concentration of 2.4 lm) were

applied Molecular mass markers were determined by

A440 (ferritin), NADH oxidation at A340 (lactate

dehy-drogenase, glutamate dehydrogenase), decomposition of

H2O2 at A240 (catalase), and ATPase activity (CSB)

Selected fractions (24–28) were upconcentrated by

spin-ning on Centricons (Millipore, Billerica, MA, USA) and

analyzed by 7% (w⁄ v) Tris ⁄ acetate SDS ⁄ PAGE and

sil-ver staining

In vitro protein–protein cross-linking

Purified recombinant CSB (60 nm) was incubated with

0.001% glutaraldehyde and 1 mm dithiothreitol in NaCl⁄ Pi

for 0, 10, 20, or 40 min at 37
C Glutaraldehyde was

quenched by adding one-tenth volumes of 1 m Tris pH 6.8,

1 m glycine Cross-linking was monitored by 3–8% (w⁄ v) Tris⁄ acetate SDS ⁄ PAGE and silver staining or western blot using the CSB antibody Dephosphorylation of CSB with protein phosphatase 1 (PP1) was performed as previously described [10]

In vivo protein–protein cross-linking Proteins were cross-linked in vivo essentially as described by Bakkenist and Kastan [29] In brief, HeLa or CSB-deficient CS1AN.S3.G2 cells were incubated with the indicated amounts of formaldehyde in minimal essential medium (In-vitrogen, Carlsbad, CA, USA) without serum for 10 min at room temperature For analysis of UV or a-amanitin influ-ence on cross-linking, HeLa cells were irradiated with 0 or

6 JÆm)2UV or incubated with 5 lm a-amanitin Cells were subsequently incubated for 4 h prior to formaldehyde (10 mm) cross-linking Formaldehyde was washed out using NaCl⁄ Pi with 100 mm glycine Nuclear extracts prepared with the NE-PER extraction kit (Pierce, Rockford, IL, USA) were analyzed by 3–8% (w⁄ v) Tris⁄ acetate SDS⁄ PAGE and western blotting using CSB and p89 anti-body (1 : 1000, H300 and S19, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA)

In vitro CSB fragment pull-down S-Protein agarose (Novagen) was equilibrated with NaCl⁄ Pi

before incubation with 5 lg of each of the five purified CSB fragments for 1.5 h at 4
C Excess fragment, and impurities were removed by washing in NaCl⁄ Pi⁄ 0.1% (v⁄ v) Tween 20, before addition of 2 lg recombinant CSB

wt protein, in NaCl⁄ Pi⁄ 0.1% (v ⁄ v) Tween 20 with

2 lgÆmL)1bovine serum albumin, 1 : 100 protease inhibitor cocktail set III (Calbiochem, San Diego, CA, USA), 0.1 mm phenylmethylsulfonyl fluoride, 5 mm MgCl2, and

5 UÆmL)1 TURBO DNase (Ambion, Austin, TX, USA) Samples were initially incubated for 15 min at 37
C and then for 16 h at 4
C The beads were washed extensively

in NaCl⁄ Pi⁄ 0.1% (v ⁄ v) Tween 20 and buffer A and dis-solved in 2· SDS loading buffer, boiled and analyzed by SDS⁄ PAGE and western using HA and HSV antibody [Y11 (1 : 2000), Santa Cruz Biotechnology, and HSV-tag monoclonal antibody (1 : 6666), Novagen]

Acknowledgements Ulla Birk Henriksen is acknowledged for excellent technical assistance Robert M Brosh Jr and Meltem Muftuoglu are thanked for critical reading of the manuscript The project was supported by the Danish Medical Research Council (22-03-0253) M.C was sup-ported by the Carlsberg Foundation

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