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Ku by phosphorylation
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ABB Archives of Biochemistry and Biophysics 470 (2008) 1–7 www.elsevier.com/locate/yabbi
Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation Alexander E. Ochem a
a,*
, Hocine Rechreche b, Doris Skopac c, Arturo Falaschi
d
International Centre for Genetic Engineering and Biotechnology, Institutional Services and Biosafety, Padriciano 99, 34012 Trieste, Italy b U.315 INSERM, 46 Bd de la Gaye, F-13009 Marseille, France c Bioallergy International, Via del Follatoio 12, Trieste, Italy d Scuola Normale Superiore di Pisa, Via della Faggiola 17/19, Pisa, Italy Received 2 October 2007, and in revised form 7 November 2007 Available online 17 November 2007
Abstract The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA doublestrand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PKcs. This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme. Ó 2007 Elsevier Inc. All rights reserved. Keywords: DNA binding; DNA-dependent ATPase; DNA-PKcs; DNA metabolism; DNA unwinding enzyme; HDH II/Ku; Protein phosphorylation; Protein dephosphorylation
DNA helicases unwind double stranded DNA (dsDNA)1 in an energy-dependent fashion to generate the single stranded DNA species (ssDNA) which serve as templates or substrate in most DNA metabolic processes [1,2]. Protein phosphorylation and dephosphorylation, acetylation and deacetylation or methylation modulate enzyme activity and may also affect the sub-cellular localization of the modified enzymes [3–5]. The DNA-dependent protein kinase of HeLa cells (DNA-PK) phosphorylates several cellular factors involved in diverse metabolic *
Corresponding author. Fax: +39 040 226555. E-mail address: [email protected] (A.E. Ochem). 1 Abbreviations used: dsDNA, double stranded DNA; ssDNA, single stranded DNA;NHEJ, non-homologous end joining; HDH II, human DNA helicase II, PP2A, protein phosphatase 2A catalytic subunit; DNAPK, DNA-dependent protein kinase complex; DSB, double-strand break. 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.11.005
processes and also plays an important role in the process of non-homologous end joining (NHEJ) in eukaryotes for the repair of DNA double-strand breaks [6] and in the process of V(D)J recombination [7]. This serine/threonine protein kinase is a complex of two components: a 370kDa catalytic subunit, DNA-PKcs and a DNA binding component of the Ku autoantigen [8]. Ku is thought to target the protein kinase complex to the DNA and is itself a heterodimer of 70- and 83-kDa subunits. The C-terminal 19-kDa domain of the 83-kDa subunit has been shown to be responsible for the recruitment of the catalytic subunit of PK to the DNA [9]. The crystal structure of Ku reveals that each subunit of the heterodimer possesses a preformed ring-shaped domain which is independent of the presence of DNA [10]. Surprisingly however, neither separate subunit is capable of independent binding to DNA in the fashion of the heterodimer, the presence of the ring-shaped
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domain notwithstanding. It appears therefore that only the simultaneous presence of both subunits guarantees the existence of a rigid open ring structure which allows the ‘‘passage’’ of the DNA. We have previously shown that, besides its well known DNA binding capacity, Ku is also endowed with an ATP-dependent DNA unwinding as well as DNA-dependent ATPase activities designated Human DNA Helicase II (HDH II) [11]. Subsequently, using the recombinant protein expressed in Escherichia coli, we dissected the in vitro activities of HDH II/Ku [12], and showed that whereas the DNA end-binding capacity of HDH II/Ku remained a prerogative of the heterodimer, the helicase activity of the molecule resided exclusively in the smaller subunit. Since the Ku autoantigen is a substrate as well as cofactor for the kinase activity of DNA-PK, we studied the effects of phosphorylation on the DNA unwinding activity of HDH II/Ku expressed in E. coli. Materials and methods Enzymes, substrates and buffers Adenosine 5 0 -triphosphate [ATP], poly[dI-dC]Æpoly[dI-dC], dsDNAcellulose resin were purchased from Sigma Corp. (St. Louis, MO, USA). T4 polynucleotide kinase and lambda protein phosphatase were purchased from New England Biolabs (Beverly, MA, USA); protein phosphatase 2A catalytic subunit (PP2A) was bought from Boehringer (Boehringer Mannheim GmbH) and wortmanin was purchased from CalbiochemNovabiochem (San Diego, CA 92121, USA).
Preparation of DNA-PKcs, native and recombinant HDH II/Ku The expression and isolation of inclusion bodies containing the recombinant separate subunits of HDH II/Ku has already been described in detail [11]. Briefly, E. coli cells (strain BL 21 (DE3)(pLysS)) transformed with the respective plasmids for the 70 or 83 kDa subunits of HDH II/Ku were grown in LB medium containing 75 mg/L ampicillin and 25 mg/L chloramphenicol with vigorous shaking to an optical density of 0.5 at 600 nm. Protein expression was induced by addition of IPTG to a final concentration of 0.4 mM and the cultures were shaken for a further 2.5 h. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris–HCl, pH 8, 2 mM EDTA). After two freeze–thaw cycles, the extremely viscous solution was fluidified by sonication and centrifuged. The pelleted inclusion bodies containing the expressed recombinant proteins were solubilized in denaturing buffer (6 M guanidinium hydrochloride, 100 mM sodium dihydrogen phosphate, 10 mM Tris–HCl, pH 8, 10 mM b-mercaptoethanol) and purified by gel filtration on Sephacryl S300 resin (Pharmacia, Uppsala, Sweden) at 25 °C in the same buffer to eliminate possible low molecular weight contaminants of bacterial origin. From 4 L of bacteria culture for each subunit of HDH II/Ku a total of 43.7 mg for the 83-kDa subunit and 53.2 mg for the 70-kDa subunit were pooled. Equimolar amounts of these two subunits were mixed and used for the renaturation process by gradual dialysis at 4 °C and subsequently for the final dsDNA affinity purification procedure following the experimental procedures previously described [11]. Both the native HDH II/Ku and DNA-PKcs were purified from HeLa nuclear extracts by a modification of the procedure described for HDH II/ Ku [11]. These two enzymes co-purified through the initial steps of ammonium sulfate fractionation (35%) and Bio Rex (Bio-Rad Lab., Hercules, CA, USA) ion exchange chromatography. However, on a strong anion exchange chromatography on Mono Q (Pharmacia Uppsala, Sweden), DNA-PKcs eluted at 0.25 M NaCl while HDH II/Ku eluted at 0.3 M
NaCl. From an initial 1.6 g of extracted proteins only 4.5 mg of proteins containing DNA-dependent protein kinase activity were pooled after this step while 15 mg of protein containing HDH II/Ku activity were recovered. The pool for native HDH II/Ku was further purified to homogeneity by dsDNA affinity chromatography as was done for the recombinant species. Enzyme purity was confirmed, as was also done for the recombinant species, by Coomassie stained SDS–PAGE gel and by Western blot analysis using the cognate rabbit polyclonal antibody [11]. The catalytic subunit of DNA-PK was purified to apparent homogeneity over a 5 ml dsDNA-cellulose resin from which it eluted at about 0.45 M NaCl. From the initial starting material extracted from HeLa cells, 0.3 mg of apparently pure DNA-PKcs was recovered after this chromatography. Enzyme purity was checked both by Coomassie blue stained SDS–PAGE and by Western blot analysis with anti DNA-PKcs monoclonal antibody (Sigma).
DNA-dependent protein kinase assay The DNA-dependent protein kinase assay measures the transfer of the c-phosphate from a donor nucleoside-triphosphate to a chosen substrate. The 10 ll reaction mixture contained 20 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM DTT, 50 mM KCl, 0.1 mM ATP containing traces of [c32P]ATP, 100 ng of poly[dI-dC]Æpoly[dI-dC] and the appropriate protein substrate. Reactions were initiated by adding 1 ll (0.5 pmol) of the kinase solution and the reaction mixtures were incubated for 30 min at 37 °C. Reactions were terminated by adding SDS–PAGE buffer (0.3 M Tris, 5% SDS, 0.05% bromophenol blue (BPB), 5% glycerol and 30 ll bmercapto-ethanol) and the products were separated by electrophoresis on a 10% denaturing gel. Protein phosphorylation was revealed by autoradiography of the dried gels and the DNA-dependent kinase activity was determined as the incorporation of c32P phosphate in the presence of poly[dI-dC]Æpoly[dI-dC] minus the incorporation in its absence. Quantitation of this enzyme activity was also achieved by cutting the dried protein bands and counting the radioactivity contained in these bands. Inhibition of the kinase activity of DNA-PKcs was achieved by including a final concentration of 10 nM of the enzyme inhibitor, wortmanin, in the standard protein kinase assay. The presence of this concentration (and also higher concentrations) of the inhibitor totally abolished the transfer of phosphate residues to the substrate by the kinase.
Protein dephosphorylation For protein dephosphorylation we used two different protein phosphatases, namely, the serine/threonine protein phosphatase 2A catalytic subunit (PP2A) and lambda protein phosphatase. Assays with PP2A contained 25 mM triethanolamine, 2 mM dithioerythritol, 0.5 mM MnCl2, 7.5 lM BSA (pH 7.6), approximately 9.8 pmol of electrophoretically pure HeLa HDH II/Ku and 4 mU of the phosphatase in a 50 ll reaction mixture, and were incubated at 37 °C. Assays with lambda phosphatase contained 50 mM Tris–HCl, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij (pH 7.5), 9.8 pmol of HeLa HDH II/Ku and 80 U of the phosphatase in 50 ll, and were incubated at 30 °C. In parallel, a negative control reaction mixture was set up in the same conditions used for the PP2A reaction but without the phosphatase. One microliters of sample aliquots were taken, at specific time intervals (up to 90 min), from each of the reaction mixtures and used in the standard DNA helicase assays to monitor the progressive effect of the dephosphorylation on the DNA unwinding activity of the helicase and to reveal the residual helicase activity in the samples.
Preparation of the helicase substrates and helicase assay The principal DNA helicase substrate used in this study was a 47-mer oligonucleotide annealed to M13 mp18 ssDNA in its central 17 nucleotides to generate a 3 0 and 5 0 double-tailed partial duplex DNA substrate. The nucleotide sequence of this 47-mer as well as the structure of the
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helicase substrate have been described and shown earlier [11]. The 10 ll helicase reaction mixture contained 20 mM Tris–HCl (pH 8), 1 mM MgCl2, 4 mM ATP, 60 mM KCl, 8 mM DTT, 4% (w/v) sucrose, 1.2 lM bovine serum albumin and the DNA substrate. About 0.005 pmol of the substrate (generally 5000 cpm) was used in each reaction mixture. After incubation with the enzyme fractions for 30 min at 37 °C, the reaction was terminated by the addition of 10 mM EDTA, 5% glycerol, 0.3% SDS and 0.3% bromophenol blue (final concentrations). The products were analyzed by electrophoresis on a 12% non-denaturing PAGE in buffer containing 90 mM Tris, 90 mM boric acid and 2 mM EDTA, and helicase activity was visualized by autoradiography of the dried gels. The quantitation of the DNA unwinding activity present in each protein fraction was determined as described earlier by excision of the bands relative to the unwound oligonucleotides and counting the contained radioactivity with a b-counter [12]. We define 1 U of DNA helicase activity as the amount of enzyme that unwinds 1% of the substrate in 1 min at 37 °C (30% in 30 min) in the linear range of enzyme concentration dependence.
Results Phosphorylation of recombinant HDH II/Ku heterodimer with purified DNA-PK Approximately 0.65 pmol of refolded recombinant HDH II/Ku heterodimer were incubated in the presence of 100 ng of poly[dI-dC]Æpoly[dI-dC] for 30 min at 37 °C with varying amounts of DNA-PKcs to determine the optimal concentration of kinase necessary to phosphorylate this amount of HDH II/Ku in vitro. As shown in Fig. 1A, optimal DNA-dependent phosphorylation of both subunits of HDH II/Ku is obtained with 0.7 pmol of DNA-PKcs (i.e. approximately equimolar proportions of the two proteins, as expected) while higher concentrations of the kinase do not show any significant increase in the phosphorylation of the substrate. Quantitation of phosphate residues incorporated in this reaction revealed that approximately 6 pmol of phosphate are incorporated into 1 pmol of HDH II/Ku, an observation consistent with the presence of six consensus sites for DNA-PK phosphorylation in the Ku heterodimer comprising two sites in the 70 kDa subunit and four such sites in the 83 kDa subunit. Several attempts to phosphorylate this helicase with various other kinases failed to show any significant incorporation of phosphate residues on the helicase, suggesting that HDH II/Ku may be a specific substrate for the catalytic subunit of DNA-PK (data not shown).
Fig. 1. Phosphorylation and its effect on the unwinding activity of rec. HDH II/Ku. (A) Phosphorylation with increasing amounts of DNA-PKcs (indicated) in the presence or absence of DNA. 0.5 lg of recombinant HDH II/Ku were phosphorylated as described in Materials and methods and analyzed on 10% SDS–PAGE. The position of each phosphorylated protein is shown. (B) DNA helicase assay with recombinant HDH II/Ku before and after phosphorylation. Lane 1, substrate alone; lanes 2 and 3, assays with 125 and 250 ng, respectively, of DNA-PKcs; lanes 4 and 5, assays with 50 and 100 ng of non-phosphorylated rec HDH II/Ku; lanes 6 and 7, assays with 50 and 100 ng of phosphorylated HDH II/Ku, respectively; lane 8, heated substrate.
greater than fivefold stimulation of the DNA unwinding activity of phosphorylated HDH II/Ku when compared to the unwinding activity of the non-phosphorylated form (Table 1). Similarly, a comparison of the time course of DNA unwinding observed for phosphorylated HDH II/ Ku with the time course recorded for an equal amount of the non-phosphorylated enzyme (Fig. 2A and B) confirmed an approximately fivefold higher rate of DNA unwinding for phosphorylated HDH II/Ku with respect to the nonphosphorylated species (Fig. 2C). Taken together, these findings suggest that during each moment of the dsDNA unwinding reaction, the phosphorylated enzyme species
The helicase activity of recombinant HDH II/Ku is stimulated upon phosphorylation We compared the DNA unwinding activity of recombinant HDH II/Ku after phosphorylation by DNA-PKcs with the activity present in the non-phosphorylated enzyme. A significant stimulation of the DNA helicase activity was observed for phosphorylated HDH II/Ku with respect to the non-phosphorylated species (Fig. 1B: compare lanes 4 and 5 with lanes 6 and 7), whereas DNA-PKcs alone showed no detectable DNA unwinding activity (lanes 2 and 3). The quantitation of these results clearly showed a
Table 1 Quantitation of DNA unwinding by HDH II/Ku before and after in vitro phosphorylation Enzyme species
Amount (ng)
% of DNA unwinding Before phosphorylation
After phosphorylation
DNA-PKcs
125 250
<2 <2
— —
HDH II/Ku
50 100
7 12
40 70
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Fig. 3. Effect of DNA-PK or wortmanin-inhibited PK on the activity of HDH II/Ku. Helicase assay with recombinant HDH II/Ku treated with 0.7 pmol DNA-PKcs and 100 lM ATP in the presence or absence of 10 nM wortmanin. After incubation for 30 min at 37 °C, equal aliquots of these reaction mixtures were assayed for DNA unwinding activity. Lane 1, substrate alone; lane 2, assay with 50 ng of HDH II/Ku; lane 3, assay with 125 ng of DNA-PKcs; lane 4, assay with 40 ng of HDH II/Ku in the presence of 10 nM wortmanin; lane 5, assay with 40 ng of HDH II/Ku treated with PKcs and ATP; lane 6, assay with 40 ng of HDH II/Ku treated with PKcs and ATP in the presence of wortmanin.
Fig. 2. Time course of DNA unwinding for phosphorylated and nonphosphorylated rec. HDH II/Ku. (A) Phosphorylated HDH II/Ku. Lane 1, substrate; lanes 2–9, assays incubated for 0, 5, 10, 20, 30, 40, 50 and 60 min, respectively; lane 10, heated substrate. (B) Non-phosphorylated HDH II/Ku. Incubation times are the same as those shown in (A). (C) Graph representation of the data in (A) and (B).
operated at a greater pace or with a greater efficiency with respect to the non-phosphorylated enzyme. The same can not be said of the other known cellular function of HDH II/Ku: its dsDNA end-binding activity. In fact, phosphorylation of the helicase by DNAPKcs did not give rise to any significant changes in the dsDNA end-binding activity of the molecule (data not shown). However, this lacking effect of phosphorylation on the dsDNA end-binding by HDH II/Ku is in perfect agreement with the observation that such cellular Ku function takes place in a totally energy-independent fashion. Stimulation of helicase activity is dependent on protein phosphorylation We verified that the observed increase in the DNA unwinding activity of phosphorylated HDH II/Ku was effectively due to the transfer of phosphate residues to the helicase by the kinase. To this effect we compared the DNA unwinding activity observed for non-phosphorylated
HDH II/Ku with the one observed for the helicase treated with DNA-PKcs (phosphorylated species) as well as with the activity of the helicase treated with wortmanin-inhibited, therefore inactive, kinase. As shown in Fig. 3, whereas a significant increase in DNA unwinding is observed for the enzyme treated with the kinase (lane 5) with respect to the non-treated helicase (lane 2), no such increase is recorded for the helicase treated with wortmanin-inhibited DNA-PKcs (lane 6). The presence of wortmanin alone showed no significant effect on the DNA unwinding activity of HDH II/Ku (lane 4), thus excluding the remote possibility that wortmanin might have inhibited the DNA unwinding activity of the helicase. We can therefore conclude that the observed stimulation of helicase activity of the kinase-treated HDH II/Ku is due to the effective phosphorylation of the enzyme. The quantitation of these results of stimulation is presented in Table 2. Dephosphorylation of HDH II/Ku decreases its DNA unwinding activity To ascertain that the enhancement of the DNA unwinding activity of HDH II/Ku was actually due to the presence of the phosphate residues, we investigated the effects of dephosphorylation on the unwinding activity of the native enzyme purified from HeLa cells, a species of this enzyme completely phosphorylated in vivo. Therefore, HeLa HDH II/Ku was treated with two different protein phosphatases alongside an appropriate negative control reaction mixture as described in Materials and methods. Several aliquots of this enzyme were taken during the dephosphorylation reaction from each reaction mixture and the residual DNA unwinding activity was compared to the unwinding activity present in the negative control reaction. Fig. 4 shows that the DNA unwinding activity of HeLa HDH II/Ku remained constant after incubation
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Table 2 Effect of wortmanin on the phosphorylation-stimulated DNA unwinding activity of HDH II/Ku Enzyme species
Amount (ng)
In vitro modification
% DNA unwinding
DNA-PKcs Recombinant HDH II/Ku DNA-PK-treated HDH II/Ku Rec. HDH II/Ku treated with kinase in the presence of wortmanin Rec. HDH II/Ku treated with wortmanin
125 100 100 100 100
— Non-phosphorylated Phosphorylated by the kinase Phosphorylation inhibited by wortmanin —
<2 13 75 13 13
Fig. 4. Effect of dephosphorylation on the DNA unwinding activity of HeLa HDH II/Ku. Protein dephosphorylation reactions were carried out as described in Materials and methods. At specific time intervals, 1-ll aliquots were taken from each reaction mixture, used in the standard DNA unwinding assay and quantitated. The graph is a comparison of the residual DNA unwinding activity in the different enzyme samples.
for 90 min with only buffer; whereas, when this enzyme was treated with a phosphatase, a significant decrease in DNA unwinding was recorded down to less than one fifth of the original rate of DNA unwinding contained in the non-treated helicase. Since the activity of phosphatases, the removal of phosphate residues, is essentially opposed to that of the kinase, we may infer that the result of this assay indirectly confirms our conclusion that phosphorylation by DNAPKcs stimulates the dsDNA unwinding activity of E. coli produced HDH II/Ku. Discussion The results obtained in this study show that, as is the case with several enzymes of DNA metabolism [13–15] the unwinding activity of HDH II/Ku can be modulated by the state of phosphorylation of the helicase. The enzymic activity of E. coli expressed HDH/Ku is stimulated upon phosphorylation, a post-translational modification not attainable in bacteria. The phosphorylated enzyme shows an approximately fivefold higher rate of DNA unwinding with respect to the non-phosphorylated species. Phosphorylation may have resulted in an improved utilisation of ATP to disrupt the hydrogen bonds in the DNA substrate, in good agreement with the report of Cao et al. [16] who observed an up-regulation of the ATPase activity of the Ku protein upon phosphorylation by DNA-PKcs. Vashisht et al. [17] have also reported the stimulation of the DNA unwinding activities of PDH47, a plant
DNA helicase isolated from pea, upon phosphorylation by protein kinase C underlining the importance of this posttranslational modification on the enzymatic activity of many eukaryotic cellular enzymes. This stimulation of unwinding activity clearly results from the effective transfer of phosphate residues to the helicase rather than by any stabilizing effect due to the presence of the kinase catalytic subunit or by an intrinsic helicase activity of this subunit (see Fig. 3, lane 3). In fact, according to the report by Chan and Lees-Miller, [18] the kinase complex dissociates into its subunits upon autophosphorylation of the catalytic subunit, thereby rendering the helicase independent of the catalytic subunit of DNA-PK. Moreover, no stimulation of DNA unwinding activity was observed when the kinase was blocked with its inhibitor. Since wortmanin alone had no detectable effect on the DNA unwinding activity of HDH II/Ku, the lack of stimulation of activity when HDH II/Ku is treated with the kinase in the presence of its inhibitor may best be explained by the failure of the kinase to phosphorylate the helicase. In vivo HDH II/Ku is totally phosphorylated, since we have observed that the HeLa cell purified enzyme is not a suitable substrate for DNA-PKcs in vitro (data not shown); hence, it is not surprising that dephosphorylation of HeLa HDH II/Ku with two different phosphatases, namely, the serine/threonine protein phosphatase 2A catalytic subunit (PP2A) and lambda protein phosphatase (the latter capable of hydrolysing phosphate groups also from tyrosine residues) caused the loss of approximately 80% of the unwinding activity originally present in the untreated enzyme, in agreement with the fivefold stimulation brought about by the phosphorylation of the non-phosphorylated species. The striking similarity between the effect of these two phosphatases would indicate that HDH II/Ku is primarily phosphorylated on serine/threonine residues. Furthermore, this observed decrease in the DNA unwinding activity of HeLa-purified HDH II/Ku consequent upon dephosphorylation of this enzyme is in agreement with the report by Douglas et al. (2001) that the activity of the kinase complex is modulated by a PP2A-like phosphatase in vivo [19]. One principal in vivo role defined for the Ku autoantigen is its involvement, as subunit of the DNA-dependent protein kinase complex (DNA-PK), in the repair of DNA double-strand breaks in non-homologous end joining (NHEJ) in eukaryotes and also in V(D)J recombination. Other studies have also reported the involvement of the DNAPK complex in other cellular functions such as RNA
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metabolism [20] and DNA replication [21] suggesting that this protein kinase complex may be involved in more cellular processes in vivo than have so far been reported. Certainly, the elucidation of the molecular mechanisms underlying these cellular processes and the specific role/s of the kinase complex therein would require more detailed studies of the same. The involvement of DNA-PK in cellular NHEJ has been extensively documented by several lines of evidence reporting deficiencies or inability in numerous phenotypes, bearing mutations in the subunits of DNAPK, to accomplish either NHEJ or V(D)J recombination, or both [7,22–25]. Although the functions of the components of this complex in these processes are still unknown, an attractive model for the participation of DNA-PK in NHEJ has been presented by Dynan et al. [26]. According to this model, the helicase activity of HDH II/Ku would be required for DNA unwinding in the search for microhomology between annealing DNA strands. In this context, our results show that the rate of DNA unwinding during this search for homology may be modulated by the state of phosphorylation of HDH II/Ku. We did not observe any effects of phosphorylation on the dsDNA end-binding activity of HDH II/Ku, a fundamental function of this component of the kinase complex required in the initial phases of the intricate process of DNA double-strand break (DSB) repair through NHEJ. This finding is not surprising firstly because the mode of binding of HDH II/Ku heterodimer to DNA, which is typified by the entry of dsDNA into the ring-shaped Ku molecule and the subsequent sliding of this protein molecule along dsDNA do not require any form of energy to be accomplished. Secondly, Douglas et al. (2005) have reported that the phosphorylation of the subunits of Ku by DNA-PKcs is not required for DSB repair even though this remains a cellular process in which the DNA-binding activity of HDH II/Ku is sine qua non and prerequisite [27], suggesting that the phosphorylation of the Ku subunits for the dsDNA binding event might represent an unnecessary dissipation of energy. Several studies have reported that the autophosphorylation of the DNA-PKcs during NHEJ repair of DSBs leads to inactivation of the kinase activity and subsequent disassembly of the kinase complex [28–30]. Other studies [31–34] have further emphasized that the removal of the components of the kinase complex following the autophosphorylation of the catalytic subunit is essential for the subsequent steps of NHEJ since the necessary DNA end-processing and efficient ligation by the other members of the repair machinery upon juxtaposition of the ligating DNA ends, would otherwise be hindered by the presence of the bulky components of the kinase complex. Taking these reports together, and in the light of the results presented in this study, it would be envisioned that in DSB repair by NHEJ, initially HDH II/Ku binds to dsDNA ends and loads the PKcs. This latter phosphorylates the helicase and enhances the unwinding activity in search of microhomology and DNA end alignment. This event is followed by the loading
and phosphorylation/activation of other members of the repair machinery by the protein kinase complex. Finally the autophosphorylation of the catalytic subunit of the kinase complex leads to the disassembly of the complex to release the DNA ends for the ligation event leading to the repair of the DNA double-strand break. Acknowledgment This project was funded with Grant No. 99.00649.PF33, Fondo Speciale MURST, of the Consiglio Nazionale delle Ricerche (CNR), Roma, Italy. References [1] S.W. Matson, K. Kaiser-Rogers, Annu. Rev. Biochem. 59 (1990) 289–329. [2] K. Geider, K. Hoffman-Berling, Annu. Rev. Biochem. 50 (1981) 233– 260. [3] D.M. Virshup, A.A.R. Russo, T.J. Kelly, Mol. Cell Biol. 12 (1992) 4883–4895. [4] R. Fotedar, J.M. Roberts, EMBO J. 11 (1992) 2177–2187. [5] B.O. Petersen, J. Lukas, J.B. Sorensen, K. Helin, EMBO J. 18 (1999) 396–410. [6] T. Carter, I. Vancurova, I. Sun, W. Lou, W.S. DeLeon, Mol. Cell Biol. 10 (1990) 6460–6471. [7] S.P. Jackson, P.A. Jeggo, TIB 20 (1995) 412–415. [8] T.M. Gottlieb, S.P. Jackson, Cell 72 (1993) 131–142. [9] B.K. Singleton, M.I. Torres-arzayus, S.T. Rottinghaus, G.E. Taccioli, P.A. Jeggo, Mol. Cell Biol. 19 (1999) 3267–3277. [10] J.R. Walker, R.A. Corpina, J. Goldberg, Nature 412 (2001) 607– 614. [11] N. Tuteja, R. Tuteja, A. Ochem, P. Taneja, N.W. Huang, A. Simoncsits, S. Susic, K. Rahman, L. Marusic, J. Chen, J. Zang, S. Wang, S. Pongor, A. Falaschi, EMBO J. 13 (1994) 4991–5001. [12] A.E. Ochem, D. Skopac, M. Costa, T. Rabilloud, L. Vuillard, A. Simoncsits, M. Giacca, A. Falaschi, J. Biol. Chem. 272 (1997) 29919– 29926. [13] P.V. Jallepalli, G.W. Brown, M. Muzi-Falconi, D. Tien, T.J. Kelly, Genes Dev. 11 (1997) 2767–2779. [14] W. Jiang, N.J. Wells, T. Hunter, Proc. Natl. Acad. Sci. USA 96 (1999) 6193–6198. [15] Y. Ishimi, Y. Komamura-Khono, J. Biol. Chem. 276 (2001) 34428– 34433. [16] Q.P. Cao, S. Pitt, J. Leszyk, J.E.F. Baril, Biochemistry 33 (1994) 8548–8557. [17] A.A. Vashisht, A. Pradhan, R. Tuteja, N. Tuteja, Plant J. 44 (2005) 76–87. [18] D.W. Chan, S. Lees-Miller, J. Biol. Chem. 271 (1996) 8936–8941. [19] P. Douglas, G.B.G. Moorhead, R. Ye, S.P. Lees-Miller, J. Biol. Chem. 276 (2001) 18992–18998. [20] S. Zhang, B. Schlott, M. Go¨rlach, F. Grosse, Nucleic Acids Res. 32 (2004) 1–10. [21] D. Matheos, M.T. Ruiz, G.B. Price, M. Zannis-Hadjopoulos, Biochem. Biophys. Acta 1578 (2002) 59–72. [22] W.K. Rathmel, G. Chu, Proc. Natl. Acad. Sci. USA 91 (1994) 7623– 7627. [23] N.V. Boubnov, D.T. Weaver, Mol. Cell Biol. 15 (1995) 5700–5706. [24] B.K. Singleton, A. Priestley, H. Steingrimsdottir, D. Gell, T. Blunt, S.P. Jackson, A.R. Lehmann, P.A. Jeggo, Mol. Cell Biol. 17 (1997) 1264–1273. [25] D.A. Ramsden, M. Gellert, EMBO J. 17 (1998) 609–614. [26] W.S. Dynan, S. Yoo, Nucleic Acids Res. 26 (1998) 1551–1559. [27] P. Douglas, S. Gupta, N. Morrice, K. Meek, S.P. Lees-Miller, DNA Repair (Amst.) 4 (2005) 1006–1018.
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[31] P. Calsou, P. Frit, O. Humbert, C. Muller, D.J. Chen, B. Salles, J. Biol. Chem. 274 (1999) 7848–7856. [32] E. Weterings, N.S. Verkaik, H.T. Bruggenwirth, J.H. Hoeijmakers, D.C. van Gent, Nucleic Acids Res. 31 (2003) 7238– 7246. [33] W.D. Block, Y. Yu, D. Merkle, J.L. Gifford, Q. Ding, K. Meek, S.P. Lees-Miller, Nucleic Acids Res. 32 (2004) 4351–4357. [34] X. Cui, Y. Yu, S. Gupta, Y.M. Cho, S.P. Lees-Miller, K. Meek, Mol. Cell Biol. 25 (2005) 10842–10852.
ABB Archives of Biochemistry and Biophysics 470 (2008) 1–7 www.elsevier.com/locate/yabbi
Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation Alexander E. Ochem a
a,*
, Hocine Rechreche b, Doris Skopac c, Arturo Falaschi
d
International Centre for Genetic Engineering and Biotechnology, Institutional Services and Biosafety, Padriciano 99, 34012 Trieste, Italy b U.315 INSERM, 46 Bd de la Gaye, F-13009 Marseille, France c Bioallergy International, Via del Follatoio 12, Trieste, Italy d Scuola Normale Superiore di Pisa, Via della Faggiola 17/19, Pisa, Italy Received 2 October 2007, and in revised form 7 November 2007 Available online 17 November 2007
Abstract The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA doublestrand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PKcs. This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme. Ó 2007 Elsevier Inc. All rights reserved. Keywords: DNA binding; DNA-dependent ATPase; DNA-PKcs; DNA metabolism; DNA unwinding enzyme; HDH II/Ku; Protein phosphorylation; Protein dephosphorylation
DNA helicases unwind double stranded DNA (dsDNA)1 in an energy-dependent fashion to generate the single stranded DNA species (ssDNA) which serve as templates or substrate in most DNA metabolic processes [1,2]. Protein phosphorylation and dephosphorylation, acetylation and deacetylation or methylation modulate enzyme activity and may also affect the sub-cellular localization of the modified enzymes [3–5]. The DNA-dependent protein kinase of HeLa cells (DNA-PK) phosphorylates several cellular factors involved in diverse metabolic *
Corresponding author. Fax: +39 040 226555. E-mail address: [email protected] (A.E. Ochem). 1 Abbreviations used: dsDNA, double stranded DNA; ssDNA, single stranded DNA;NHEJ, non-homologous end joining; HDH II, human DNA helicase II, PP2A, protein phosphatase 2A catalytic subunit; DNAPK, DNA-dependent protein kinase complex; DSB, double-strand break. 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.11.005
processes and also plays an important role in the process of non-homologous end joining (NHEJ) in eukaryotes for the repair of DNA double-strand breaks [6] and in the process of V(D)J recombination [7]. This serine/threonine protein kinase is a complex of two components: a 370kDa catalytic subunit, DNA-PKcs and a DNA binding component of the Ku autoantigen [8]. Ku is thought to target the protein kinase complex to the DNA and is itself a heterodimer of 70- and 83-kDa subunits. The C-terminal 19-kDa domain of the 83-kDa subunit has been shown to be responsible for the recruitment of the catalytic subunit of PK to the DNA [9]. The crystal structure of Ku reveals that each subunit of the heterodimer possesses a preformed ring-shaped domain which is independent of the presence of DNA [10]. Surprisingly however, neither separate subunit is capable of independent binding to DNA in the fashion of the heterodimer, the presence of the ring-shaped
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domain notwithstanding. It appears therefore that only the simultaneous presence of both subunits guarantees the existence of a rigid open ring structure which allows the ‘‘passage’’ of the DNA. We have previously shown that, besides its well known DNA binding capacity, Ku is also endowed with an ATP-dependent DNA unwinding as well as DNA-dependent ATPase activities designated Human DNA Helicase II (HDH II) [11]. Subsequently, using the recombinant protein expressed in Escherichia coli, we dissected the in vitro activities of HDH II/Ku [12], and showed that whereas the DNA end-binding capacity of HDH II/Ku remained a prerogative of the heterodimer, the helicase activity of the molecule resided exclusively in the smaller subunit. Since the Ku autoantigen is a substrate as well as cofactor for the kinase activity of DNA-PK, we studied the effects of phosphorylation on the DNA unwinding activity of HDH II/Ku expressed in E. coli. Materials and methods Enzymes, substrates and buffers Adenosine 5 0 -triphosphate [ATP], poly[dI-dC]Æpoly[dI-dC], dsDNAcellulose resin were purchased from Sigma Corp. (St. Louis, MO, USA). T4 polynucleotide kinase and lambda protein phosphatase were purchased from New England Biolabs (Beverly, MA, USA); protein phosphatase 2A catalytic subunit (PP2A) was bought from Boehringer (Boehringer Mannheim GmbH) and wortmanin was purchased from CalbiochemNovabiochem (San Diego, CA 92121, USA).
Preparation of DNA-PKcs, native and recombinant HDH II/Ku The expression and isolation of inclusion bodies containing the recombinant separate subunits of HDH II/Ku has already been described in detail [11]. Briefly, E. coli cells (strain BL 21 (DE3)(pLysS)) transformed with the respective plasmids for the 70 or 83 kDa subunits of HDH II/Ku were grown in LB medium containing 75 mg/L ampicillin and 25 mg/L chloramphenicol with vigorous shaking to an optical density of 0.5 at 600 nm. Protein expression was induced by addition of IPTG to a final concentration of 0.4 mM and the cultures were shaken for a further 2.5 h. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris–HCl, pH 8, 2 mM EDTA). After two freeze–thaw cycles, the extremely viscous solution was fluidified by sonication and centrifuged. The pelleted inclusion bodies containing the expressed recombinant proteins were solubilized in denaturing buffer (6 M guanidinium hydrochloride, 100 mM sodium dihydrogen phosphate, 10 mM Tris–HCl, pH 8, 10 mM b-mercaptoethanol) and purified by gel filtration on Sephacryl S300 resin (Pharmacia, Uppsala, Sweden) at 25 °C in the same buffer to eliminate possible low molecular weight contaminants of bacterial origin. From 4 L of bacteria culture for each subunit of HDH II/Ku a total of 43.7 mg for the 83-kDa subunit and 53.2 mg for the 70-kDa subunit were pooled. Equimolar amounts of these two subunits were mixed and used for the renaturation process by gradual dialysis at 4 °C and subsequently for the final dsDNA affinity purification procedure following the experimental procedures previously described [11]. Both the native HDH II/Ku and DNA-PKcs were purified from HeLa nuclear extracts by a modification of the procedure described for HDH II/ Ku [11]. These two enzymes co-purified through the initial steps of ammonium sulfate fractionation (35%) and Bio Rex (Bio-Rad Lab., Hercules, CA, USA) ion exchange chromatography. However, on a strong anion exchange chromatography on Mono Q (Pharmacia Uppsala, Sweden), DNA-PKcs eluted at 0.25 M NaCl while HDH II/Ku eluted at 0.3 M
NaCl. From an initial 1.6 g of extracted proteins only 4.5 mg of proteins containing DNA-dependent protein kinase activity were pooled after this step while 15 mg of protein containing HDH II/Ku activity were recovered. The pool for native HDH II/Ku was further purified to homogeneity by dsDNA affinity chromatography as was done for the recombinant species. Enzyme purity was confirmed, as was also done for the recombinant species, by Coomassie stained SDS–PAGE gel and by Western blot analysis using the cognate rabbit polyclonal antibody [11]. The catalytic subunit of DNA-PK was purified to apparent homogeneity over a 5 ml dsDNA-cellulose resin from which it eluted at about 0.45 M NaCl. From the initial starting material extracted from HeLa cells, 0.3 mg of apparently pure DNA-PKcs was recovered after this chromatography. Enzyme purity was checked both by Coomassie blue stained SDS–PAGE and by Western blot analysis with anti DNA-PKcs monoclonal antibody (Sigma).
DNA-dependent protein kinase assay The DNA-dependent protein kinase assay measures the transfer of the c-phosphate from a donor nucleoside-triphosphate to a chosen substrate. The 10 ll reaction mixture contained 20 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM DTT, 50 mM KCl, 0.1 mM ATP containing traces of [c32P]ATP, 100 ng of poly[dI-dC]Æpoly[dI-dC] and the appropriate protein substrate. Reactions were initiated by adding 1 ll (0.5 pmol) of the kinase solution and the reaction mixtures were incubated for 30 min at 37 °C. Reactions were terminated by adding SDS–PAGE buffer (0.3 M Tris, 5% SDS, 0.05% bromophenol blue (BPB), 5% glycerol and 30 ll bmercapto-ethanol) and the products were separated by electrophoresis on a 10% denaturing gel. Protein phosphorylation was revealed by autoradiography of the dried gels and the DNA-dependent kinase activity was determined as the incorporation of c32P phosphate in the presence of poly[dI-dC]Æpoly[dI-dC] minus the incorporation in its absence. Quantitation of this enzyme activity was also achieved by cutting the dried protein bands and counting the radioactivity contained in these bands. Inhibition of the kinase activity of DNA-PKcs was achieved by including a final concentration of 10 nM of the enzyme inhibitor, wortmanin, in the standard protein kinase assay. The presence of this concentration (and also higher concentrations) of the inhibitor totally abolished the transfer of phosphate residues to the substrate by the kinase.
Protein dephosphorylation For protein dephosphorylation we used two different protein phosphatases, namely, the serine/threonine protein phosphatase 2A catalytic subunit (PP2A) and lambda protein phosphatase. Assays with PP2A contained 25 mM triethanolamine, 2 mM dithioerythritol, 0.5 mM MnCl2, 7.5 lM BSA (pH 7.6), approximately 9.8 pmol of electrophoretically pure HeLa HDH II/Ku and 4 mU of the phosphatase in a 50 ll reaction mixture, and were incubated at 37 °C. Assays with lambda phosphatase contained 50 mM Tris–HCl, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij (pH 7.5), 9.8 pmol of HeLa HDH II/Ku and 80 U of the phosphatase in 50 ll, and were incubated at 30 °C. In parallel, a negative control reaction mixture was set up in the same conditions used for the PP2A reaction but without the phosphatase. One microliters of sample aliquots were taken, at specific time intervals (up to 90 min), from each of the reaction mixtures and used in the standard DNA helicase assays to monitor the progressive effect of the dephosphorylation on the DNA unwinding activity of the helicase and to reveal the residual helicase activity in the samples.
Preparation of the helicase substrates and helicase assay The principal DNA helicase substrate used in this study was a 47-mer oligonucleotide annealed to M13 mp18 ssDNA in its central 17 nucleotides to generate a 3 0 and 5 0 double-tailed partial duplex DNA substrate. The nucleotide sequence of this 47-mer as well as the structure of the
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helicase substrate have been described and shown earlier [11]. The 10 ll helicase reaction mixture contained 20 mM Tris–HCl (pH 8), 1 mM MgCl2, 4 mM ATP, 60 mM KCl, 8 mM DTT, 4% (w/v) sucrose, 1.2 lM bovine serum albumin and the DNA substrate. About 0.005 pmol of the substrate (generally 5000 cpm) was used in each reaction mixture. After incubation with the enzyme fractions for 30 min at 37 °C, the reaction was terminated by the addition of 10 mM EDTA, 5% glycerol, 0.3% SDS and 0.3% bromophenol blue (final concentrations). The products were analyzed by electrophoresis on a 12% non-denaturing PAGE in buffer containing 90 mM Tris, 90 mM boric acid and 2 mM EDTA, and helicase activity was visualized by autoradiography of the dried gels. The quantitation of the DNA unwinding activity present in each protein fraction was determined as described earlier by excision of the bands relative to the unwound oligonucleotides and counting the contained radioactivity with a b-counter [12]. We define 1 U of DNA helicase activity as the amount of enzyme that unwinds 1% of the substrate in 1 min at 37 °C (30% in 30 min) in the linear range of enzyme concentration dependence.
Results Phosphorylation of recombinant HDH II/Ku heterodimer with purified DNA-PK Approximately 0.65 pmol of refolded recombinant HDH II/Ku heterodimer were incubated in the presence of 100 ng of poly[dI-dC]Æpoly[dI-dC] for 30 min at 37 °C with varying amounts of DNA-PKcs to determine the optimal concentration of kinase necessary to phosphorylate this amount of HDH II/Ku in vitro. As shown in Fig. 1A, optimal DNA-dependent phosphorylation of both subunits of HDH II/Ku is obtained with 0.7 pmol of DNA-PKcs (i.e. approximately equimolar proportions of the two proteins, as expected) while higher concentrations of the kinase do not show any significant increase in the phosphorylation of the substrate. Quantitation of phosphate residues incorporated in this reaction revealed that approximately 6 pmol of phosphate are incorporated into 1 pmol of HDH II/Ku, an observation consistent with the presence of six consensus sites for DNA-PK phosphorylation in the Ku heterodimer comprising two sites in the 70 kDa subunit and four such sites in the 83 kDa subunit. Several attempts to phosphorylate this helicase with various other kinases failed to show any significant incorporation of phosphate residues on the helicase, suggesting that HDH II/Ku may be a specific substrate for the catalytic subunit of DNA-PK (data not shown).
Fig. 1. Phosphorylation and its effect on the unwinding activity of rec. HDH II/Ku. (A) Phosphorylation with increasing amounts of DNA-PKcs (indicated) in the presence or absence of DNA. 0.5 lg of recombinant HDH II/Ku were phosphorylated as described in Materials and methods and analyzed on 10% SDS–PAGE. The position of each phosphorylated protein is shown. (B) DNA helicase assay with recombinant HDH II/Ku before and after phosphorylation. Lane 1, substrate alone; lanes 2 and 3, assays with 125 and 250 ng, respectively, of DNA-PKcs; lanes 4 and 5, assays with 50 and 100 ng of non-phosphorylated rec HDH II/Ku; lanes 6 and 7, assays with 50 and 100 ng of phosphorylated HDH II/Ku, respectively; lane 8, heated substrate.
greater than fivefold stimulation of the DNA unwinding activity of phosphorylated HDH II/Ku when compared to the unwinding activity of the non-phosphorylated form (Table 1). Similarly, a comparison of the time course of DNA unwinding observed for phosphorylated HDH II/ Ku with the time course recorded for an equal amount of the non-phosphorylated enzyme (Fig. 2A and B) confirmed an approximately fivefold higher rate of DNA unwinding for phosphorylated HDH II/Ku with respect to the nonphosphorylated species (Fig. 2C). Taken together, these findings suggest that during each moment of the dsDNA unwinding reaction, the phosphorylated enzyme species
The helicase activity of recombinant HDH II/Ku is stimulated upon phosphorylation We compared the DNA unwinding activity of recombinant HDH II/Ku after phosphorylation by DNA-PKcs with the activity present in the non-phosphorylated enzyme. A significant stimulation of the DNA helicase activity was observed for phosphorylated HDH II/Ku with respect to the non-phosphorylated species (Fig. 1B: compare lanes 4 and 5 with lanes 6 and 7), whereas DNA-PKcs alone showed no detectable DNA unwinding activity (lanes 2 and 3). The quantitation of these results clearly showed a
Table 1 Quantitation of DNA unwinding by HDH II/Ku before and after in vitro phosphorylation Enzyme species
Amount (ng)
% of DNA unwinding Before phosphorylation
After phosphorylation
DNA-PKcs
125 250
<2 <2
— —
HDH II/Ku
50 100
7 12
40 70
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Fig. 3. Effect of DNA-PK or wortmanin-inhibited PK on the activity of HDH II/Ku. Helicase assay with recombinant HDH II/Ku treated with 0.7 pmol DNA-PKcs and 100 lM ATP in the presence or absence of 10 nM wortmanin. After incubation for 30 min at 37 °C, equal aliquots of these reaction mixtures were assayed for DNA unwinding activity. Lane 1, substrate alone; lane 2, assay with 50 ng of HDH II/Ku; lane 3, assay with 125 ng of DNA-PKcs; lane 4, assay with 40 ng of HDH II/Ku in the presence of 10 nM wortmanin; lane 5, assay with 40 ng of HDH II/Ku treated with PKcs and ATP; lane 6, assay with 40 ng of HDH II/Ku treated with PKcs and ATP in the presence of wortmanin.
Fig. 2. Time course of DNA unwinding for phosphorylated and nonphosphorylated rec. HDH II/Ku. (A) Phosphorylated HDH II/Ku. Lane 1, substrate; lanes 2–9, assays incubated for 0, 5, 10, 20, 30, 40, 50 and 60 min, respectively; lane 10, heated substrate. (B) Non-phosphorylated HDH II/Ku. Incubation times are the same as those shown in (A). (C) Graph representation of the data in (A) and (B).
operated at a greater pace or with a greater efficiency with respect to the non-phosphorylated enzyme. The same can not be said of the other known cellular function of HDH II/Ku: its dsDNA end-binding activity. In fact, phosphorylation of the helicase by DNAPKcs did not give rise to any significant changes in the dsDNA end-binding activity of the molecule (data not shown). However, this lacking effect of phosphorylation on the dsDNA end-binding by HDH II/Ku is in perfect agreement with the observation that such cellular Ku function takes place in a totally energy-independent fashion. Stimulation of helicase activity is dependent on protein phosphorylation We verified that the observed increase in the DNA unwinding activity of phosphorylated HDH II/Ku was effectively due to the transfer of phosphate residues to the helicase by the kinase. To this effect we compared the DNA unwinding activity observed for non-phosphorylated
HDH II/Ku with the one observed for the helicase treated with DNA-PKcs (phosphorylated species) as well as with the activity of the helicase treated with wortmanin-inhibited, therefore inactive, kinase. As shown in Fig. 3, whereas a significant increase in DNA unwinding is observed for the enzyme treated with the kinase (lane 5) with respect to the non-treated helicase (lane 2), no such increase is recorded for the helicase treated with wortmanin-inhibited DNA-PKcs (lane 6). The presence of wortmanin alone showed no significant effect on the DNA unwinding activity of HDH II/Ku (lane 4), thus excluding the remote possibility that wortmanin might have inhibited the DNA unwinding activity of the helicase. We can therefore conclude that the observed stimulation of helicase activity of the kinase-treated HDH II/Ku is due to the effective phosphorylation of the enzyme. The quantitation of these results of stimulation is presented in Table 2. Dephosphorylation of HDH II/Ku decreases its DNA unwinding activity To ascertain that the enhancement of the DNA unwinding activity of HDH II/Ku was actually due to the presence of the phosphate residues, we investigated the effects of dephosphorylation on the unwinding activity of the native enzyme purified from HeLa cells, a species of this enzyme completely phosphorylated in vivo. Therefore, HeLa HDH II/Ku was treated with two different protein phosphatases alongside an appropriate negative control reaction mixture as described in Materials and methods. Several aliquots of this enzyme were taken during the dephosphorylation reaction from each reaction mixture and the residual DNA unwinding activity was compared to the unwinding activity present in the negative control reaction. Fig. 4 shows that the DNA unwinding activity of HeLa HDH II/Ku remained constant after incubation
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Table 2 Effect of wortmanin on the phosphorylation-stimulated DNA unwinding activity of HDH II/Ku Enzyme species
Amount (ng)
In vitro modification
% DNA unwinding
DNA-PKcs Recombinant HDH II/Ku DNA-PK-treated HDH II/Ku Rec. HDH II/Ku treated with kinase in the presence of wortmanin Rec. HDH II/Ku treated with wortmanin
125 100 100 100 100
— Non-phosphorylated Phosphorylated by the kinase Phosphorylation inhibited by wortmanin —
<2 13 75 13 13
Fig. 4. Effect of dephosphorylation on the DNA unwinding activity of HeLa HDH II/Ku. Protein dephosphorylation reactions were carried out as described in Materials and methods. At specific time intervals, 1-ll aliquots were taken from each reaction mixture, used in the standard DNA unwinding assay and quantitated. The graph is a comparison of the residual DNA unwinding activity in the different enzyme samples.
for 90 min with only buffer; whereas, when this enzyme was treated with a phosphatase, a significant decrease in DNA unwinding was recorded down to less than one fifth of the original rate of DNA unwinding contained in the non-treated helicase. Since the activity of phosphatases, the removal of phosphate residues, is essentially opposed to that of the kinase, we may infer that the result of this assay indirectly confirms our conclusion that phosphorylation by DNAPKcs stimulates the dsDNA unwinding activity of E. coli produced HDH II/Ku. Discussion The results obtained in this study show that, as is the case with several enzymes of DNA metabolism [13–15] the unwinding activity of HDH II/Ku can be modulated by the state of phosphorylation of the helicase. The enzymic activity of E. coli expressed HDH/Ku is stimulated upon phosphorylation, a post-translational modification not attainable in bacteria. The phosphorylated enzyme shows an approximately fivefold higher rate of DNA unwinding with respect to the non-phosphorylated species. Phosphorylation may have resulted in an improved utilisation of ATP to disrupt the hydrogen bonds in the DNA substrate, in good agreement with the report of Cao et al. [16] who observed an up-regulation of the ATPase activity of the Ku protein upon phosphorylation by DNA-PKcs. Vashisht et al. [17] have also reported the stimulation of the DNA unwinding activities of PDH47, a plant
DNA helicase isolated from pea, upon phosphorylation by protein kinase C underlining the importance of this posttranslational modification on the enzymatic activity of many eukaryotic cellular enzymes. This stimulation of unwinding activity clearly results from the effective transfer of phosphate residues to the helicase rather than by any stabilizing effect due to the presence of the kinase catalytic subunit or by an intrinsic helicase activity of this subunit (see Fig. 3, lane 3). In fact, according to the report by Chan and Lees-Miller, [18] the kinase complex dissociates into its subunits upon autophosphorylation of the catalytic subunit, thereby rendering the helicase independent of the catalytic subunit of DNA-PK. Moreover, no stimulation of DNA unwinding activity was observed when the kinase was blocked with its inhibitor. Since wortmanin alone had no detectable effect on the DNA unwinding activity of HDH II/Ku, the lack of stimulation of activity when HDH II/Ku is treated with the kinase in the presence of its inhibitor may best be explained by the failure of the kinase to phosphorylate the helicase. In vivo HDH II/Ku is totally phosphorylated, since we have observed that the HeLa cell purified enzyme is not a suitable substrate for DNA-PKcs in vitro (data not shown); hence, it is not surprising that dephosphorylation of HeLa HDH II/Ku with two different phosphatases, namely, the serine/threonine protein phosphatase 2A catalytic subunit (PP2A) and lambda protein phosphatase (the latter capable of hydrolysing phosphate groups also from tyrosine residues) caused the loss of approximately 80% of the unwinding activity originally present in the untreated enzyme, in agreement with the fivefold stimulation brought about by the phosphorylation of the non-phosphorylated species. The striking similarity between the effect of these two phosphatases would indicate that HDH II/Ku is primarily phosphorylated on serine/threonine residues. Furthermore, this observed decrease in the DNA unwinding activity of HeLa-purified HDH II/Ku consequent upon dephosphorylation of this enzyme is in agreement with the report by Douglas et al. (2001) that the activity of the kinase complex is modulated by a PP2A-like phosphatase in vivo [19]. One principal in vivo role defined for the Ku autoantigen is its involvement, as subunit of the DNA-dependent protein kinase complex (DNA-PK), in the repair of DNA double-strand breaks in non-homologous end joining (NHEJ) in eukaryotes and also in V(D)J recombination. Other studies have also reported the involvement of the DNAPK complex in other cellular functions such as RNA
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metabolism [20] and DNA replication [21] suggesting that this protein kinase complex may be involved in more cellular processes in vivo than have so far been reported. Certainly, the elucidation of the molecular mechanisms underlying these cellular processes and the specific role/s of the kinase complex therein would require more detailed studies of the same. The involvement of DNA-PK in cellular NHEJ has been extensively documented by several lines of evidence reporting deficiencies or inability in numerous phenotypes, bearing mutations in the subunits of DNAPK, to accomplish either NHEJ or V(D)J recombination, or both [7,22–25]. Although the functions of the components of this complex in these processes are still unknown, an attractive model for the participation of DNA-PK in NHEJ has been presented by Dynan et al. [26]. According to this model, the helicase activity of HDH II/Ku would be required for DNA unwinding in the search for microhomology between annealing DNA strands. In this context, our results show that the rate of DNA unwinding during this search for homology may be modulated by the state of phosphorylation of HDH II/Ku. We did not observe any effects of phosphorylation on the dsDNA end-binding activity of HDH II/Ku, a fundamental function of this component of the kinase complex required in the initial phases of the intricate process of DNA double-strand break (DSB) repair through NHEJ. This finding is not surprising firstly because the mode of binding of HDH II/Ku heterodimer to DNA, which is typified by the entry of dsDNA into the ring-shaped Ku molecule and the subsequent sliding of this protein molecule along dsDNA do not require any form of energy to be accomplished. Secondly, Douglas et al. (2005) have reported that the phosphorylation of the subunits of Ku by DNA-PKcs is not required for DSB repair even though this remains a cellular process in which the DNA-binding activity of HDH II/Ku is sine qua non and prerequisite [27], suggesting that the phosphorylation of the Ku subunits for the dsDNA binding event might represent an unnecessary dissipation of energy. Several studies have reported that the autophosphorylation of the DNA-PKcs during NHEJ repair of DSBs leads to inactivation of the kinase activity and subsequent disassembly of the kinase complex [28–30]. Other studies [31–34] have further emphasized that the removal of the components of the kinase complex following the autophosphorylation of the catalytic subunit is essential for the subsequent steps of NHEJ since the necessary DNA end-processing and efficient ligation by the other members of the repair machinery upon juxtaposition of the ligating DNA ends, would otherwise be hindered by the presence of the bulky components of the kinase complex. Taking these reports together, and in the light of the results presented in this study, it would be envisioned that in DSB repair by NHEJ, initially HDH II/Ku binds to dsDNA ends and loads the PKcs. This latter phosphorylates the helicase and enhances the unwinding activity in search of microhomology and DNA end alignment. This event is followed by the loading
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