Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein

Functional consequences of mutations in theconserved SF2 motifs and post-translationalphosphorylation of the CSB proteinMette Christiansen, Tinna Stevnsner, Charlotte Modin1, Pia M. Martensen1,

Robert M. Brosh Jr2 and Vilhelm A. Bohr2,*

Danish Center for Molecular Gerontology and 1Department of Molecular Biology, University of Aarhus,DK-8000 Aarhus C, Denmark and 2Laboratory of Molecular Gerontology, National Institute on Aging, NationalInstitutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA

Received October 9, 2002; Revised and Accepted November 15, 2002

ABSTRACT

The rare inherited human genetic disorderCockayne syndrome (CS) is characterized by devel-opmental abnormalities, UV sensitivity and prema-ture aging. The cellular and molecular phenotypesof CS include increased sensitivity to UV-inducedand oxidative DNA lesions. Two genes are involved:CSA and CSB. The CS group B (CSB) protein hasroles in transcription, transcription-coupled repair,and base excision repair. It is a DNA stimulatedATPase and remodels chromatin in vitro. Here, wehave analyzed wild-type (wt) and motif II, V and VImutant CSB proteins. We ®nd that the mutantproteins display different degrees of ATPase activityde®ciency, and in contrast to the in vivo comple-mentation studies, the motif II mutant is moredefective than motif V and VI CSB mutants.Furthermore, CSB wt ATPase activity was studiedwith different biologically important DNA cofactors:DNA with different secondary structures and dam-aged DNA. The results indicate that the state of DNAsecondary structure affects the level of CSB ATPaseactivity. We ®nd that the CSB protein is phosphory-lated in untreated cells and that UV irradiation leadsto its dephosphorylation. Importantly, dephosphory-lation of the protein in vitro results in increasedATPase activity of the protein, suggesting that theactivity of the CSB protein is subject to phosphory-lation control in vivo. These observations may havesigni®cant implications for the function of CSBin vivo.

INTRODUCTION

Nucleotide excision repair (NER) is a complicated process,which removes a broad spectrum of DNA lesions. Twosubpathways of NER exist: (i) the global genome repair

pathway and (ii) the transcription-coupled repair (TCR)pathway, and TCR is an important factor in Cockaynesyndrome (CS) (1). CS is a premature aging syndrome withcomplex symptoms, including developmental abnormalities,neurologic dysfunction and short average life span. Cellularcharacteristics include hypersensitivity to UV light, andfailure of RNA synthesis to recover to normal rates followingUV irradiation. Two genes have been shown to be involved:CSA and CSB (2). The 168 kDa CS group B (CSB) proteincontains an acidic amino acid stretch, a glycine-rich region,two putative nuclear localization signal (NLS) sequences, andseven conserved ATPase motifs (3). Members of the SNF2-like family of DNA-dependent ATPases, including CSB,contain seven characteristic motifs, I, Ia and II±VI, that arealso present in DNA and RNA helicases (4). Helicase activityhas not been demonstrated for any members of the SNF2-likefamily, which is part of Superfamily 2 (SF2), but in generalthey have the ability to destabilize protein±DNA interactions(5). The CSB protein displays DNA-dependent ATPaseactivity, which is better stimulated by double-stranded DNA(dsDNA) than single-stranded DNA (ssDNA) (6±8), and CSBis able to remodel chromatin in vitro (9).

So far, only helicases, and thus no proteins of the SNF2-likefamily, have been crystallized. However, structures derivedfrom the crystallized SF1 helicases Rep and PcrA revealstriking similarity to the distantly related SF2 RNA helicaseNS3, and the seven conserved helicase motifs form anucleotide binding (NTB) pocket (10). In particular, theconserved aspartate and glutamate residues of motif II(Walker B) coordinate the Mg2+ ion and probably also theattacking water molecule during ATP hydrolysis (11,12).Motif V seems to contact the oligonucleotide, while motif VIis directly or indirectly involved in NTP binding (11,12).

The integrity of the SNF2-like ATPase domain is critical formost functions of CSB in vivo. CSB cDNA with pointmutations in motifs Ia, II, III, V and VI, as opposed to wild-type (wt) CSB cDNA, do not complement the de®ciencies ofthe SV40-transformed CS-B cell line, CS1AN.S3.G2 (13,14).In contrast, both a deletion of the acidic region and a pointmutation in a putative NTB motif do not interfere with theability of CSB to complement CSB-de®cient cells (14±16).

*To whom correspondence should be addressed. Tel: +1 410 558 8562; Fax: +1 410 558 8157; Email: [email protected]

Nucleic Acids Research, 2003, Vol. 31, No. 3 963±973DOI: 10.1093/nar/gkg164

CS-B cells and cell extracts are generally de®cient inincision at 8-oxoguanine (8-oxoG) and 8-oxoadenine lesions(13,17±19). The reduced 8-oxoG activity can be comple-mented by transfection with wt CSB and partially comple-mented by motif II (E646Q) mutant cDNA, but not with motifV (T912/913V) or VI (Q942E) CSB mutants (13,20).Furthermore, the 8-oxoG repair capacity of wt and mutanttransfected cells correlates with mRNA and protein levels ofthe 8-oxoG DNA glycosylase (OGG1) responsible for theremoval of 8-oxoG (17,18). This suggests that the general baseexcision repair (BER) defect is associated with a subtletranscription de®ciency, and that wt and motif II but not motifV and VI mutant CSB proteins are able to support ef®cientOGG1 expression. There also appears to be a functionalinteraction between OGG1 and CSB (21). To better under-stand the biological functions of the mutant CSB proteins, wereport here the functional analysis of puri®ed, recombinantCSB wt and motif II, V and VI mutant proteins. We ®nd thatthe ATPase activity of the motif II CSB mutant is nearlyabolished whereas motif V and VI mutant proteins retainresidual catalytic activity. DNA binding of CSB mutantproteins is similar to the CSB wt protein. The motif V and VImutant proteins show slightly reduced ATP binding while themotif II mutant protein exhibits increased ATP binding. Also,CSB wt and mutant protein tagged with enhanced cyano¯uorescent protein (ECFP) all display homogenous nucleardistribution. These results suggest that de®ciencies in func-tions other than DNA binding or DNA-stimulated ATPhydrolysis detected in vitro may be responsible for the defectsof CS-B null cell lines transfected with either motif V or motifVI CSB mutant genes. Furthermore, CSB wt ATPase activitywas studied with different biologically important DNAcofactors, and the results indicate that the CSB wt ATPaseactivity is maximally stimulated by DNA substrates contain-ing a small loop or bubble.

Although the CSB protein is involved in RNA polymeraseII (RNAPII) elongation, it does not affect the transcription ofall genes, suggesting that the activity of the CSB protein isregulated in vivo (13,22). Control of phosphorylation status isan important factor in both transcription and repair (23±25),and phosphorylation affects subcellular localization as well asactivity of different members of the SNF2 family (26,27).Phosphorylation of target proteins is an integral part of theDNA damage response, and the p53 protein, which interactswith CSB, is phosphorylated in response to DNA damage(28,29). The extent of phosphorylation of the CSB protein hasnot previously been investigated and we were interested in thisparameter and, particularly, how phosphorylation status mightaffect its activities and how it might change during DNArepair. Here we present evidence that CSB activity iscontrolled by phosphorylation. Notably, we document thatCSB ATPase activity is stimulated by dephosphorylation, andthat UV irradiation results in dephosphorylation of CSBin vivo.

MATERIALS AND METHODS

Plasmid constructs

CSB wt containing an N-terminal hemagglutinin antigen (HA)epitope and a C-terminal histidine (His6) cloned in the

pFASTBAC vector, pFB-CSBwt (8), was kindly providedby Alan Lehmann. A P¯M1 fragment of pcDNA3.1-CSBcontaining mutations resulting in either E646Q, T912/913V orQ942E substitutions (13,14), was exchanged with thecorresponding fragment in pFB-CSBwt using the rapidligation kit according to the manufacturer's instructions(Roche), thus generating pFB-CSBE646Q, pFB-CSBT912/913V and pFB-CSBQ942E.

Baculovirus production and infection

Bacmids and virus from pFB-CSBwt, pFB-CSBE646Q,pFB-CSBT912/913V and pFB-CSBQ942E were producedusing the BAC to BAC Baculovirus Overexpression System(LifeTechnologies, Inc.) essentially as described in theaccompanying manual. Suspension cultures of HiFive cellswere infected with the recombinant baculoviruses at amultiplicity of infection of 10. Three days post-infectioncells were collected from a 200 ml culture and washed twice inice-cold phosphate-buffered saline (PBS).

CSB protein puri®cation

Recombinant proteins were puri®ed using a modi®cation of amethod described by Citterio et al. (8). Pellet was lysedin ice-cold lysis buffer 1 (25 mM Tris±HCl pH 9.0, 1 mMEDTA, 10% glycerol, 1% NP-40, 0.3 M KCl, 1 mMb-mercaptoethanol, 0.1 mM PMSF, 2 mM each of chymosta-tin, leupeptin, antipain, pepstatin A). The lysate wascentrifuged at 12000 g for 15 min. For puri®cation the®ltrated supernatant was diluted with 2 vol buffer 2 (buffer 1 atpH 6.8 and without NP-40 and EDTA). A Ni2+±nitriloaceticacid±agarose column (Qiagen) equilibrated with buffer A(25 mM HEPES±KOH pH 7, 0.01% NP-40, 10% glycerol,1 mM b-mercaptoethanol, 0.1 mM PMSF, 0.3 M KCl) and5 mM imidazole was loaded with the diluted lysate. Thecolumn was washed with buffer A and 20 mM imidazole.Proteins were eluted with buffer A and a 20 ml linear gradientof 20±250 mM imidazole and CSB eluted at ~60 mMimidazole. A HiTrap heparin column (Amersham PharmaciaBiotech) equilibrated with buffer A was directly loaded withthe CSB containing Ni2+±nitriloacetic acid fractions. Thecolumn was washed with buffer A, and the proteins wereeluted with buffer A and 0.8 M KCl. Protein was stored inaliquots at ±80°C. Protein concentration was determined in the¯ow-through, wash and selected fractions of both theNi2+±nitriloacetic acid±agarose column and the heparin col-umn by Bradford analysis. Purity of CSB was checked by 7%acrylamide Tris±acetate SDS±PAGE (Invitrogen) and byimmunoblotting using a Penta-His antibody (Qiagen).Furthermore, speci®c activity of different Ni2+±nitriloaceticagarose fractions and heparin fractions was tested, and theactivity corresponded well with the pooled fractions.

CSB ATPase activity

Standard reactions (10 ml) were performed with 150 ng ofDNA cofactor, supercoiled (>90%) pUC19 plasmid, 10 ng ofprotein, and 1 mCi of [g-32P]ATP (3000 Ci/mmol; HartmannAnalytic) in buffer B (20 mM Tris±HCl pH 7.5, 4 mM MgCl2,

50 mM ATP, 40 mg/ml BSA, 1 mM DTT). Reactionswere incubated for 1 h at 30°C and stopped by the additionof 5 ml of 0.5 M EDTA. Samples (1 ml) were analyzedon a polyethylenimine-cellulose thin layer chromatography

964 Nucleic Acids Research, 2003, Vol. 31, No. 3

(PEI-TLC) plate developed in 0.75 M KH2PO4. Plates wereexposed on screen and ATP hydrolysis was analyzed using aMolecular Imager. For activity determinations, either theamount of protein or DNA cofactor was varied. For deter-mination of kinetic parameters the amount of CSB protein waskept constant, while the amount of cold substrate was variedbetween 5 and 100 mM, or ATP concentration was keptconstant and the time was varied (5, 10, 20, 30, 40, 60 min).Less than 20% of the ATP was hydrolyzed during theincubations. Results from multiple experiments are reported asmean values with standard deviations. Differences wereassessed by one-way analysis of variance followed byStudent's t-test, and P < 0.05 was considered to be signi®cant.

Cofactor DNA substrates

Initially, pUC19 plasmid was puri®ed using Qiagen maxiprep(protocol supplied by manufacturer). CsCl-gradient analysisshowed that >90% was in a supercoiled form.

UV damage. The pUC19 plasmid was irradiated with 300 J/m2

on ice resulting in approximately 10 lesions per plasmid (30).

Methylene blue (MB). Fifty micrograms of plasmid DNA werediluted with 0.30 ml of H2O and 0.3 ml of 6 mM MBwas added. The diluted sample was irradiated with visiblelight (18.7 kJ/m2/s) for 3 min, resulting in approximately 108-oxoG per plasmid (31).

Cisplatin. Forty micrograms of pUC19 plasmid were incu-bated overnight at 37°C with 4.8 mM cisplatin in 3 mM NaCl,

0.5 mM Na2HPO4, 0.5 mM NaH2PO4, resulting in 10 adductsper plasmid (32). The reaction was stopped by addition ofNaCl to 60 mM.

Methyl methane sulfonate (MMS). Ten micromolar MMS with100 mM DNA (~1000 bp) in 10 mM Tris pH 7.8 givesapproximately 1 adduct per 1000 nt (33). pUC19 (40 mg) wasincubated overnight at 37°C with 0.48 mM MMS and 10 mMTris pH 7.8.

The DNA was precipitated, washed, redissolved and dilutedto 150 ng/ml, and checked on an agarose gel. The syntheticoligonucleotides used (DNA Technology, Denmark), wereFPLC-puri®ed, and are shown in Table 1.

DNA binding assay

A 166 bp DdeI pUC19 fragment was labeled using the Klenowfragment (Invitrogen) and 5 mCi [a-32P]ATP (300 mCi/mmol;Amersham Pharmacia Biotech). Nitrocellulose ®lters werepre-wetted in binding buffer (25 mM HEPES±KOH pH 8.3,40 mM MgCl2, 10 mM EDTA, 450 mg/ml BSA, 10 mM DTT,10% glycerol) and indicated amounts of CSB proteins wereincubated with 1 ng of DNA in binding buffer with or without3 mM ATP-gS or ADP. Samples were incubated for 30 min atroom temperature, and 100 ml of binding buffer was added.Samples were passed through a nitrocellulose ®lter, washedtwice with 0.5 ml of binding buffer, and exposed on a screen.Background radioactivity bound in the absence of protein wassubtracted from total radioactivity bound to the ®lter andpercent bound DNA of total DNA was determined. A Hill plotwas used to analyze the data (34).

Table 1. DNA substrates used as cofactors in ATPase assays

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ATP binding assay

CSB wt and mutant proteins (25, 50, 100 and 200 nM) wereincubated with 10 nM DNA, 83 nM ATP-g35S in buffer B for5 min at 30°C. Samples were applied to a pre-wettednitrocellulose ®lter (13 buffer B without BSA) and washed103 with ATP wash buffer (50 mM Tris pH 7.9, 0.1 M EDTA,5 mM MgCl2, 2.5% glycerol, 800 mM KCl). Filters wereexposed to screen. Background radioactivity bound in theabsence of protein was subtracted from total radioactivitybound to the ®lter and the data from at least three independentexperiments were normalized against results obtained for25 nM CSB wt protein.

Localization of ECFP-tagged CSB

CSB wt cDNA was cloned upstream of the ECFP gene inpMC-ECFP (kindly provided by Christian Mielke), which wasconstructed from pMC-2P (35), to give rise to the vector pMC-CSB-ECFP. The ECFP-tagged CSB E646Q, Q942E andT912/913V mutant vectors were produced by replacing the1300 bp NsiI fragment of pMC-CSB-ECFP with each of themutant cDNA sequences obtained by PCR ampli®cation frompcDNA3.1-CSB-derived plasmids containing the respectivemutations (13,14). All PCR-ampli®ed sequences were veri®edby full-length sequencing. CS1AN.S3.G2 cells, grown inDMEM supplemented with 10% fetal calf serum (FCS)(Gibco BRL, Life Technologies), 100 U/ml penicillin,and 100 mg/ml streptomycin, were transfected with theresulting plasmids using Lipofectamine (Gibco BRL, LifeTechnologies) according to the manufacturer's instructions.Stably transfected cell lines were selected and maintained inmedium containing 0.2 mg/ml puromycin. pMC-CSB-ECFP-transfected cells were further selected for UV resistance bytreatment with 4 J/m2 UV-C on 3 consecutive days. Thecellular distribution of ECFP-tagged proteins was analyzedemploying an epi¯uorescence microscope (Olympus) andpictures were captured using a Cooled CCD camera.

In vitro phosphorylation of CSB

Whole-cell extracts for in vitro phosphorylation were preparedby lysing 2±5 3 106 cells with radioimmunoprecipitation(RIPA) buffer (150 mM NaCl, 1% Triton, 0.1% SDS, 10 mMTris pH 8, 0.5% sodium deoxycholate, 0.2 mM PMSF,20 mg/ml aprotinin, 10 mg/ml leupeptine, 1 mM Na-orthovanadate, 10 U/ml DNase) supplemented with phospha-tase inhibitors (Sigma, 1:100). The lysate was microcentri-fuged at 16 000 g for 10 min at 4°C. In vitro phosphorylationreactions were carried out with kinase buffer (20 mMTris±HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, 25 mM ATP)and 5 mCi of [g-32P]ATP. GTP, which is also a phosphoryldonor of casein kinase II (CKII), was added when indicated.The reactions (10 ml) were initiated by the addition of CKII(New England Biolabs) or whole-cell extracts and carried outat 30°C for 15 min. The resulting products were analyzed byelectrophoresis on a 7% acrylamide Tris±acetate gel(Invitrogen). The gel was washed and visualized by aMolecular Imager. Alternatively, phosphorylation reactionswere performed without [g-32P]ATP, and the products ana-lyzed by immunoblotting.

Dephosphorylation of CSB

CSB protein was incubated with the indicated amount ofprotein phosphatase 1 (PP1; New England Biolabs) inphosphatase buffer (50 mM Tris±HCl pH 7.0 at 25°C,0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35, 1 mM MnCl2and 3 mM ATP) for 15 min at 30°C. The products wereanalyzed by immunoblotting or used in standard ATPasereactions as described above.

In vivo phosphorylation of CSB

Human diploid ®broblasts, GM38B, were seeded and grownfor 48 h. Cells (2±5 3 106) were incubated with phosphate-free DMEM (Gibco) supplemented with 5% dialyzed FCS for1 h to exhaust the pool of endogenous phosphate, and thenirradiated with 0 or 6 J/m2 UV. The cells were supplementedwith fresh phosphate-free medium containing 100 mCi 32Pinorganic phosphate (Pi) (Hartmann Analytic) for 4 h.Radiolabeling was terminated by washing twice with ice-cold PBS, cells were lysed with RIPA buffer, and the lysatewas centrifuged at 12 000 g for 10 min at 4°C. The supernatantwas pre-cleared with Protein A magnetic beads (New EnglandBiolabs), and incubated with 5 mg of polyclonal anti-ERCC6(A. F. SchuÈtzdeller, Germany) for 1 h at 4°C. Protein Amagnetic beads were incubated with immune complexes for 1h at 4°C, and beads were collected using the accompanyingmagnet. The beads were washed three times with RIPA bufferand proteins were analyzed by SDS±PAGE and visualized bya Molecular Imager and silver stain.

RESULTS

CSB wt and mutant protein activity

Site-directed mutations in ATPase motifs II, V and VI(Fig. 1A) were subcloned into the pFB-CSBwt vector, thusenabling puri®cation of these mutants as recombinantproteins. CSB cDNA containing any of these mutations, incontrast to wt CSB, cannot complement most of the defects ofCS1AN.S3.G2 cells (Fig. 1B) (13,20). Figure 2A shows thatthe CSB wt and CSB mutant proteins were of similar purity asseen by silver staining, and had the expected size of ~168 kDa.The introduced mutations are minor conserved substitutions,which are not expected to exhibit signi®cant structuralperturbations. We observed that CSB wt and mutant proteinsshowed the same behavior in solution and during chroma-tography and aggregated at high concentrations in solution.

The CSB protein is a DNA-stimulated ATPase, withpreference for dsDNA (8), and CSB wt protein puri®ed inour laboratory showed the same behavior (Fig. 2B). A single-stranded pre-melted 60mer stimulated the CSB ATPaseactivity weakly when compared to a double-stranded 60meror plasmid DNA, whereas very little or no activity was seenwith a (dT)16 oligonucleotide or no DNA. In all subsequentATPase assays, 50 mM cold ATP was included to keep theATP hydrolysis below 20% and thereby allow activitydeterminations. The CSB ATPase shows a clear dose responseupon addition of increasing amounts of pUC19 cofactor DNA(Fig. 2C). CSB ATPase activity showed a plateau above 50 ngof pUC19 DNA (Fig. 2C). Thus, to ensure that the ATPaseactivity is not limited by availability of cofactor DNA, 150 ngof DNA was used in subsequent reactions. Lineweaver±Burke


analysis of the data yielded a Keffector value for pUC19 DNA of3.1 6 0.9 nM (55 6 16 ng).

The extent of ATP hydrolysis was observed to be propor-tional to the amount of CSB wt present (Fig. 3). Theconservative substitution E646Q in motif II of the SNF2-likedomain resulted in complete abolishment of the ATPasefunction of the protein. In contrast, it was seen that the twoother conservative substitutions T912/913V and Q942E inmotifs V and VI, respectively, retained a low level of ATPaseactivity (Fig. 3). Since the CSB wt and mutant proteins werepuri®ed by the same procedure and are at a similar level ofpurity and concentration (Fig. 2A), the ATPase activity is notdue to any contaminating species, but is a property of thepuri®ed proteins. Furthermore, we performed simultaneousanalysis of the speci®c activity, purity of CSB wt and mutantproteins, and the amount of CSB present in the ¯ow-through,wash and different fractions of both the Ni2+±nitriloaceticacid±agarose column and the heparin column. This analysisshowed that the differences in activity are not due to loss ofactive enzyme during the puri®cation procedure (data notshown). The protein titrations are also consistent with animpairment of ATPase activity of the mutant proteins, andeven large quantities of mutant enzyme, compared to wt, donot overcome the de®ciency.

Km was calculated in ®ve independent experiments withconstant protein concentration, supercoiled pUC19 plasmidand an ATP concentration ranging from 5 to 100 mM. A Km

value for ATP of ~13 mM with dsDNA as the cofactor and aKm of ~23 mM with ssDNA was calculated for the CSB wtprotein (Table 2). kcat was calculated from ATPase kineticsanalysis with time ranging from 5 to 60 min. kcat was ~31 min±1

with dsDNA as the cofactor and with ssDNA cofactor kcat was~5 min±1 for the CSB wt protein (Table 2), and these resultsare quite similar to results reported earlier (6,8). CSBQ942Ehas a 4.6-fold increase in Km accompanied by a 3.8-folddecrease in kcat compared to CSB wt protein. The CSBT912/913V protein has a 9.2-fold increase in Km and, as the

CSBQ942E protein, a 3.8-fold reduction in kcat whencompared to CSB wt. These data indicate that mutations ofconserved residues in motifs V and VI impair ATP binding aswell as ATP hydrolysis itself.

In addition to the ATPase activity, we also examined ATPand DNA binding of the different mutant CSB proteins, usinga nitrocellulose ®lter-binding assay. In support of the kinetic

Figure 1. Diagram showing the CSB protein. (A) The recombinant CSBprotein contains a HA epitope; a highly acidic region, A; a glycine richregion, G; two serine phosphorylation signals, S; preceded by NLSs, L; theseven conserved Swi/Snf ATPase motifs, I, IA, II±VI; a NTB fold, NTB;and ®nally the C-terminal histidine tag, His. Introduced N- and C-terminaltags are shown in gray. Site-directed mutations in ATPase motifs II, V andVI are indicated. (B) Complementation characteristics of the wt and mutantproteins in CS1AN.S3.G2 cells (13,14,18).

Figure 2. CSB puri®cation and ATPase activity. (A) Comparison of 100 ngof each of the puri®ed proteins by silver stain and immunoblot using Hisantibody. wt, CSB wt; E, CSBE646Q; Q, CSBQ942E; T, CSBT912/913V.(B) [g-32P]ATP was incubated with 10 ng of recombinant CSB wt protein inthe presence or absence of the indicated DNA cofactors under standardreaction conditions but without 50 mM cold ATP (see Materials andMethods), and analyzed on PEI-TLC. (C) Quanti®cation of ATP hydrolysisafter incubation with 10 ng of recombinant CSB wt, 50 mM cold ATP, andthe indicated amounts of supercoiled, undamaged pUC19 plasmid DNA.Release of Pi was quanti®ed using a Molecular Imager. Error bars representstandard deviations of at least three independent experiments.

Figure 3. ATPase activity of CSB wt and mutant proteins. [g-32P]ATPhydrolysis after incubation with 0±150 ng of recombinant CSB wt andmutant proteins, 50 mM cold ATP and 150 ng of plasmid DNA under stand-ard reaction conditions. Error bars represent standard deviations of at leastthree independent experiments.


data, ATP binding of the motif V and VI mutants was reducedcompared to the CSB wt protein (Fig. 4, data are normalizedagainst ATP binding of 25 nM CSB wt protein). In contrast,the motif II mutant exhibited increased ATP binding com-pared to CSB wt protein (Fig. 4). The DNA binding analysisshowed that CSB wt protein exhibited DNA binding in theabsence of nucleotide and reduced DNA binding in thepresence of ADP or the weakly hydrolyzable ATP analogATPgS (Fig. 5A). To determine the apparent dissociationconstant, the data were analyzed by a Hill plot. In the absenceof nucleotide, Kd was 0.27 mM, while the presence of ATPgSincreased Kd to 1.5 mM. In the presence of ADP, Kd was foundto be 2.9 mM. Puri®ed CSB mutant proteins all exhibitedDNA binding with Kd at wt levels both in the absence (Fig. 5B)and presence of nucleotide (data not shown).

Localization of CSB wt and mutant proteins

To determine whether differences in in vivo complementationdata could be attributed to a de®ciency in subcellularlocalization, which may arise as a result of misfolding dueto the amino acid substitutions, CSB wt and mutant proteinswere tagged with ECFP and expressed in CS1AN.S3.G2 cells.The analysis showed that ECFP alone localized all over thecell, whereas the CSB wt-ECFP fusion protein localized to thenucleus and rescued the CS1AN cells from UV sensitivity(Fig. 6, and data not shown). ECFP fusions with the three

different mutants also showed predominant nuclear localiza-tion (Fig. 6). The tagged CSB proteins were homogeneouslydistributed in the nucleus with ~20% of the cells showingadditional brighter ¯uorescent foci.

CSB ATPase activity dependence on secondarystructures of the DNA cofactor and on substratescontaining DNA damage

It was next investigated whether CSB ATPase activity isaffected by cofactor DNA substrates with different secondarystructure related to NER or transcription arrest (bubble of15 nt), or transcription elongation (bubble of 35 nt) (36,37).The structures tested are shown in Table 1. Interestingly, theATPase activity was signi®cantly increased (1.6-fold) with

Table 2. Kinetic parameters of CSB wt and mutant ATPase

CSB wt CSBE646Q CSBQ942E CSBT912/913VThis study Selby and Sancar (22) Citterio et al. (8) This study This study This study

ATPase kcat (min±1)dsDNA 31 6 6 45±53 27±33 nd 8 6 2 8 6 3ssDNA 5.1 6 0.6 3.7±6

ATPase Km (mM)dsDNA 13 6 1 nd 60 6 10 120 6 20ssDNA 23 6 5

DNA effectors were supercoiled pUC19 plasmid (dsDNA) or preboiled single-stranded 60mer (ssDNA). For kcat determinations, 10 ng of CSB, 50 mM ATPand 150 ng of DNA were used and the time of incubation was varied (0, 5, 10, 20, 30, 40 and 60 min). For Km determinations, 10 ng of CSB, 150 ng ofDNA, and varying concentrations of ATP (5±100 mM) were incubated for 1 h. Values represent mean 6 standard deviations of at least three independentexperiments; nd, Pi release was undetectable, thus no parameters could be determined.

Figure 4. ATP binding of CSB wt and mutant proteins. CSB wt protein wasincubated with ATP-g35S (83 nM). Samples were applied to nitrocellulosemembranes, washed and exposed on screen. Binding was analyzed on aMolecular Imager and data were normalized against 25 nM CSB wt. Errorbars represent standard deviations of at least three independent experiments.

Figure 5. DNA binding of CSB wt and mutant proteins. (A) Indicatedamounts of CSB wt protein with or without 3 mM ATPgS or ADP wasincubated with ~1 ng of radioactively labeled 166 bp DdeI pUC19 fragmentfor 30 min, bound to a nitrocellulose ®lter, and bound DNA was analyzedon a Molecular Imager. Points represent the mean of three independentexperiments. (B) CSB wt and mutant proteins without nucleotide were incu-bated as indicated above. Points represent the mean of three independentexperiments.


DNA cofactor substrates containing a loop of 16 thymines andwith bubble substrates containing 15 thymines in the bubble(loop and b15 in Fig. 7A). In contrast, cofactor DNAcontaining double-strand (ds) to single-strand (ss) transitionsin non-rigid structures [ds±ss and splayed arm (sa) and abubble of 35 thymines (b35)] did not increase the CSBATPase activity signi®cantly above that of a double-stranded31mer. To test if DNA damage directly can stimulate the CSBATPase, pUC19 plasmid DNA was damaged by UV irradi-ation, photoactivated MB, cisplatin or MMS. No damagedDNA had an effect, yet a consistent, but statisticallyinsigni®cant, stimulation was observed with cisplatin-treatedDNA (Fig. 7B). Since the activity of other SNF2-like familymembers is affected by the presence of interacting proteins(5), we also investigated whether CSB ATPase activity wasin¯uenced by some of its known interaction partners.However, neither XPA nor p53 had any effect on CSBATPase activity (data not shown).

Phosphorylation of the CSB protein in vitro

When the CSB gene was ®rst cloned, it was reported to harbortwo putative CKII serine phosphorylation sites in the vicinityof putative NLSs (3). It has not previously been shownwhether CSB is post-translationally modi®ed by phosphory-lation. We performed a database search, which resulted in theidenti®cation of several other possible CKII phosphorylation

sites, a number of protein kinase C phosphorylation sites andtwo tyrosine kinase phosphorylation sites. We then investi-gated whether the CSB protein is phosphorylated. Initially,recombinant CSB wt protein was incubated with puri®ed CKIIin the presence of [g-32P]ATP, and the products were analyzedby SDS±PAGE. As seen in the top panel in Figure 8A, CKII isable to phosphorylate CSB in vitro. When cold GTP, which isa substrate for the CKII kinase and not for other kinases, wasincluded in the reaction, the radioactive labeling of CSB wasdiminished (compare lanes 2±4, top panel, in Fig. 8B). Thebottom panel shows the Coomassie stain of the gel and veri®esequal loading of the CSB protein. In addition, whole-cellextracts prepared from human primary ®broblasts,GM38B cells, could phosphorylate CSB, and this phosphory-lation was out competed by addition of cold GTP as well(compare lanes 6±8, top panel, Fig. 8B), thus substantiatingthe potential role of CKII.

CSB is phosphorylated in vivo

Recombinant proteins puri®ed from insect cells are oftenphosphorylated. Western blotting and phospho-speci®c anti-bodies were used to test whether this is also the case for theCSB protein. The results shown in Figure 9A indicate thatpuri®ed recombinant CSB wt was heavily phosphorylated atthreonine residues but not at serine or tyrosine. Incubationwith CKII increased the threonine phosphorylation slightly,but also induced phosphorylation at tyrosine and serine(Fig. 9A, lanes 2 and 3). This is somewhat in con¯ict withthe substrate speci®city of CKII, which mainly phosphorylatesthreonine and serine residues, and could be explained by

Figure 6. Cellular localization of ECFP-tagged CSB wt and mutant protein.CS1AN.S3.G2 cells stably transfected with N-terminal fusion of ECFP toCSB wt and mutant proteins. Images were captured using epi¯uorescencemicroscopy.

Figure 7. CSB ATPase activity stimulated by different cofactor DNAsubstrates. Reaction conditions were as described in Figure 2 with 150 ngDNA. (A) CSB wt ATPase activity in the presence of synthetic oligonucle-otides with the indicated secondary structures. (B) CSB wt ATPase activityin the presence of pUC19 DNA damaged with UV radiation: MB, methy-lene blue and visible light; CP, cisplatin; MMS, methyl methane sulfonate,as described in Materials and Methods. Values were normalized againstcontrol DNA; *P < 0.05, statistically signi®cantly different from controlDNA.


cross-reactivity of the antibody. Furthermore, it was tested ifPP1 was able to remove the phosphorylation of therecombinant CSB. As shown in Figure 9A, lanes 4 and 5,PP1 reduced CSB phosphorylation signi®cantly.

We next analyzed CSB phosphorylation in human cellsin vivo. Normal human diploid ®broblasts (GM38B) wereradioactively labeled with inorganic phosphate, proteins wereisolated and CSB was precipitated by the use of a CSB-speci®c antibody. The lower panels of Figure 9B show thesilver staining of precipitated proteins analyzed bySDS±PAGE, and indicate that similar amounts of CSB wereprecipitated in untreated cells and cells irradiated with 6 J/m2.CSB is clearly phosphorylated in unirradiated cells (toppanels, Fig. 9B) and, interestingly, UV radiation abolishesCSB phosphorylation after 4 h, while phosphorylation of CSBis partially recovered 24 h after UV (Fig. 9, results werecon®rmed in three independent biological experiments).

CSB ATPase activity is regulated by phosphorylation

Having concluded that CSB is phosphorylated in vivo, we nextinvestigated whether phosphorylation might affect the cata-lytic activity of CSB. ATPase activity was analyzed afterdephosphorylating the recombinant CSB protein. As shown inFigure 10, dephosphorylation with 100 mU of PP1 signi®-cantly (P < 0.01) increased the ATPase activity of the puri®edprotein by 38%. PP1 had no ATPase activity and heat-inactivated (HI) PP1 did not result in a similar increase,con®rming that increased CSB ATPase activity was a result ofdephosphorylation. We were also curious to see if the ATPaseactivity of the mutant proteins was altered by dephosphoryla-tion and found that it increased by ~40% as a result ofdephosphorylation (data not shown). However, compared toCSB wt protein, the activity remained very low for the motif Vand VI mutants and almost undetectable for the motif IImutant after dephosphorylation (data not shown).

DISCUSSION

In this study, puri®ed CSB wt, motif II, motif V and motif VIrecombinant mutant proteins were characterized. In motif IIthe conserved glutamate was substituted with glutamine

Figure 10. CSB ATPase activity is increased after dephosphorylation.Recombinant CSB wt protein (100 ng) was incubated with 50, 100 mU ofPP1 or 100 mU of HI PP1. After dephosphorylation, 1/10 of the reaction(10 ng of CSB) was transferred to ATPase reactions and incubated for afurther 1 h. Error bars represent standard deviations of at least threeindependent experiments and values were normalized against untreatedCSB. *P < 0.01, statistically signi®cantly different from untreated CSB.

Figure 9. Phosphorylation of the CSB protein in vivo. (A) Puri®edrecombinant CSB wt protein (1 mg) was phosphorylated with 50 or 200 Uof CKII or dephosphorylated with 100 or 500 mU of PP1. After immuno-blotting, phosphorylation was detected using phosphospeci®c antibodies asindicated. (B) CSB is phosphorylated in vivo. GM38B cells were irradiatedeither with 0 or 6 J/m2 and incubated for 4 or 24 h with 32P Pi phosphate at15 mCi/ml. The cells were lysed and immunoprecipitated with antibodyagainst CSB. Immunoprecipitated proteins were resolved using 7% acryla-mide Tris±acetate SDS±PAGE. The gel was washed and visualized with aMolecular Imager. Lower panel shows the silver staining of the same gel tocon®rm that equal amounts of protein were loaded. Arrow points to CSBprotein; size (in kDa) of a protein marker is indicated.

Figure 8. Phosphorylation of the CSB protein in vitro. (A) RecombinantCSB wt protein was incubated with 50 U of puri®ed CKII in the presenceof [g-32P]ATP for 0, 10 or 20 min. CSB phosphorylation was detected bySDS±PAGE, exposure on screen and the use of a Molecular Imager.Coomassie stain of the same gel indicates equal loading. (B) As (A) withthe indicated amounts of either puri®ed CKII, or whole-cell extract (WCE)and GTP as indicated.


(CSBE646Q), in motif V the two conserved threonines weresubstituted with valines (CSBT912/913V) and, ®nally, theconserved glutamine of motif VI was substituted withglutamate (CSBQ942E). Citterio et al. (8) reported thatdsDNA is the preferred DNA effector for the CSB wt proteinand our results are consistent with this. We found a kcat of~31 min±1 with dsDNA and ssDNA effector only supportedCSB ATP hydrolysis to a level of ~5 min±1.

The ATPase activity of the motif II E646Q mutant studiedhere was undetectable. This is comparable to a K538R motif Imutant reported to be completely devoid of ATPase activity(8). These results are consistent with a role of the lysine ofmotif I and the glutamate of motif II in ATP binding andhydrolysis deduced from the structural analysis of the SF1 andSF2 proteins crystallized so far (10). Also, a mutation in theeIF4A RNA helicase, corresponding to the E646Q mutation inCSB, abolished the ATPase activity of eIF4A completely (38).Despite the similar reduction in ATPase activity, RNAsynthesis recovery of CS-B cells was partially complementedby the motif I mutant but not the motif II mutant (8,13). Thedifference may reside in the types of assays used forquantifying RNA synthesis recovery [grain count of CSBmicroinjected cells versus 3H-uridine incorporation in CSB-transfected cells (8,13)]. However, it may also suggest thatwhile the ATPase activity of the two mutant proteins wasreduced to a similar extent, the two motifs do not displaytotally overlapping functions in vivo.

In contrast to motif I and II mutants, the CSBT912/913Vand CSBQ942E mutants showed residual ATPase activitywith ~4-fold reduction of kcat in the presence of dsDNA as thecofactor, and the kinetic data indicate impairment of both ATPhydrolysis and binding (Fig. 3 and Table 2). In support of this,a ®lter-binding analysis of the non-hydrolyzable ATP analogATPg35S also showed reduced ATP binding by the motif Vand VI mutant proteins (Fig. 5). Furthermore, DNA binding ofthe mutant proteins was at the wt level (Fig. 6). These resultsare comparable to results obtained for mutational analysis oftwo different virus RNA helicases. A motif V mutant plumpox potyvirus RNA helicase (T305S resembling theCSBT912/913V mutant studied here) displayed signi®cantlyreduced ATPase activity and abrogated RNA helicase activity,while RNA binding remained intact (39). Similarly, a motif VI(Q1486H) mutation of the NS3 helicase was shown toabrogate ATPase and helicase activity completely, whilesingle-stranded RNA binding was increased (40). TheCSBQ942E and CSBT912/913V mutants are very comparableto vector alone in genetic complementation of survival, RNAsynthesis recovery and apoptosis after UV treatment and alsosensitivity toward 4-nitroquinoline-1-oxide (14). Thus, thedata presented here suggest that residual ATP hydrolysis doesnot result in even partial complementation, and that ef®cientATP hydrolysis is an essential requirement for CSB tofunction in vivo in TCR and RNA synthesis recovery. Thedifferential requirement of motif II (15) and motifs V and VI(21) in 8-oxoG repair can thus not be explained by theirinvolvement in either ATP hydrolysis or DNA binding, butmust reside in other functions such as the ability to changeDNA topology or interactions with other proteins. In addition,we found similar nuclear localization of both CSB wt andmutant proteins. Therefore, we can conclude that the

complementation defects of the mutant proteins are notcaused by a de®ciency in localization.

To investigate the regulation of CSB protein activity, westudied the effect of different cofactor DNA substrates on CSBwt ATPase activity. We ®nd that substrates containing a loopor a small bubble stimulate CSB ATPase activity signi®cantly,while no increase is observed with substrates where thedouble-strand to single-strand transition is sterically lessstrained. This suggests that the CSB ATPase activity isstimulated by a sharp bend in the DNA backbone. Althoughthese data are in contrast to previous ®ndings of stimulation bya splayed arm cofactor (8), the results presented here wereobtained under conditions of <20% ATP hydrolysis, therebyallowing an actual kinetic analysis. RNAPII stalling leads totranscription bubble retraction (37), and it could be speculatedthat CSB encounters this DNA substrate when present in astalled RNAPII complex and that the CSB ATPase activity issubsequently stimulated.

The presence of DNA damage per se did not stimulate theCSB ATPase activity above that of undamaged DNA,however, cisplatin-treated DNA consistently resulted inincreased ATPase activity although not statistically signi®-cant. This may be explained by the damage spectra offered bythese different agents. The main cisplatin lesion, d(GpG) 1,2-intrastrand cross-links, induces a very prominent bend of theDNA helix (41). In contrast, the UV induced cis±syncyclobutane pyrimidine dimers and (6±4) photoproducts(42), 8-oxoG, 4,6-diamino-5-formamidopyrimidine and 2,6-diamino-4-hydroxy-5-formaidopyrimidine lesions induced byphotoactivated MB (43), and methylated bases induced byMMS (33), result in less bending of the DNA helix. Thus, theincrease in ATPase activity may be related to the degree ofhelix distortion introduced by the cisplatin lesions, and we arecurrently investigating this possibility in further detail.

The response to DNA damage involves the regulation ofprotein activity by phosphorylation of a large number ofdownstream targets. In addition, phosphorylation of the SNF2-like family proteins hBRM and BRG-1 leads to exclusion ofthe proteins from condensed chromatin during mitosis,whereas phosphorylated ATRX, which is also a SNF2-likefamily member, associates with condensed chromatin (26,27).Thus, we decided to analyze the potential phosphorylation ofthe CSB protein. Post-translational modi®cation has notpreviously to our knowledge been investigated for the CSBprotein and could play a major role in its function andregulation. Interestingly, CKII seems to play a role in the DNAdamage checkpoint. CKII cDNA overexpression partiallycomplements the UV sensitivity of xeroderma pigmentosumgroup D and C cells, and mutation of a subunit of the yeasthomolog of CKII results in de®cient adaptation to double-strand breaks and consequently irreversible arrest (44,45). We®nd that both puri®ed CKII and human cell extracts canphosphorylate recombinant CSB protein in vitro, and that GTPinhibits this phosphorylation. Furthermore, puri®ed recombi-nant CSB protein is heavily phosphorylated. Importantly, theCSB protein is found to be phosphorylated in vivo in humancells. Four hours after UV irradiation, CSB phosphorylation isabsent, while CSB phosphorylation approaches initial levels24 h after UV. This indicates that dephosphorylation of theprotein occurs during the DNA repair process (NER).


Control of phosphorylation status seems to be essential toachieve competent repair. Inhibitors of protein phosphatasescause a dramatic reduction of repair activity in cell extracts(46), i.e. excessive phosphorylation can inhibit repair. Insupport of this, the cdk-activating kinase subcomplex ofTFIIH inhibits repair in the presence of an ATP-generatingsystem (25). Furthermore, chromatin remodeling, which CSBis known to be involved in (9), can also be regulated byphosphorylation. Sif et al. (47) reported that a mitoticallyinactivated SWI/SNF complex containing phosphorylatedBRG-1 is de®cient in chromatin remodeling, and regainsnucleosome remodeling activity upon dephosphorylationin vitro. Accordingly, we observed that CSB dephosphoryla-tion in vitro resulted in an increase in ATPase activity. Ourcombined observations therefore suggest that CSB activityin vivo may also be controlled by phosphorylation, and thatCSB activity is activated during the NER process viadecreased phosphorylation. We are currently investigatinghow phosphorylation affects the chromatin remodeling func-tion of CSB, and the interaction of CSB with different NERproteins. Extracts from UV-irradiated CS cells were shown tolack hypophosphorylated, initiation competent RNAPII (48).This may indicate that both CSB and stalled RNAPII aredephosphorylated upon UV irradiation in wt cells, and thatCSB is important for the release of hypophosphorylatedRNAPII for transcription initiation.

In summary, the analyzed residues of motifs II, V and VIare essential for CSB ATPase activity as well as biologicalactivity and residual ATPase activity is insuf®cient tocomplement the UV-sensitive phenotypes of CS cells.Results presented here indicate that CSB senses DNAsecondary structure and thereby is activated. As a model ofCSB function in elongation, it can be speculated that whenRNAPII is stalled reversibly at intrinsic signals, due to RNAsecondary structure or low nucleotide pools, CSB may helppush the polymerase past the impediment as a result of DNAstructure-dependent activation of CSB. CSB may stimulateelongation both by changing the conformation of nascentDNA and by acting on chromatin structure in the proximity ofthe polymerase (9). However, when the polymerase cannot bestimulated to elongate, due to the nature of the blockage,TFIIH is recruited, the presence of damage is veri®ed andNER (or possibly BER) proceeds. We ®nd that CSB isdephosphorylated in response to UV and that dephosphoryla-tion increases CSB ATPase activity. This suggests thatphosphorylation status is involved in controlling the level ofCSB activity in vivo. Whether CSB phosphorylation iscontrolled in a cell-cycle-dependent manner, after oxidativedamage, and perhaps by RNAPII stalling are importantquestions that await further studies.

ACKNOWLEDGEMENTS

Ulla Henriksen and Inger Bjùrndal are acknowledged forexcellent technical assistance. Morten Sunesen is thanked forhis help on CSB localization studies. Jean-Philippe Laine andJeff Stuart are thanked for their critical reading of themanuscript. The project was supported by Danish ResearchCouncil, and The European Community (QLK6-CT-1999-02002).

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Documents

Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein