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ISSN 0006-2979, Biochemistry (Moscow), 2012, Vol. 77, No. 6, pp. 679-688. © Pleiades Publishing, Ltd., 2012. Phosphatase Activity in Barley Proteins Tightly Bound to DNA and Its Development-Dependent Changes K. Bielskiene. 1, D. Labeikyte. 2, N. Sjakste3,4, L. Bagdoniene. 2*, and B. Juodka2 1Laboratory of Molecular Oncology, Institute of Oncology, Vilnius University, P. Baublio 3b, Vilnius LT-08406, Lithuania 2Department of Biochemistry and Biophysics, Vilnius University, M. K. Ciurlionio 21, Vilnius LT-03101, Lithuania, fax: 370-5-239-8231; E-mail: [email protected]; [email protected] 3Faculty of Medicine, University of Latvia, Sarlotes 1a, Riga LV1001, Latvia 4Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV 1006, Latvia Received February 29, 2012 Revision received March 11, 2012 Abstract—The tightly bound proteins (TBPs), a protein group that remains attached to DNA either covalently or noncova- lently after deproteinization, have been found in numerous eukaryotic species. Some TBPs isolated from mammalian and yeast cells possess phosphatase or kinase activity. The aim of this study was to characterize further TBPs in barley (Hordeum vulgare) cells. The spectra of TBPs varied in different organs of barley shoots (first leaves, coleoptile, and roots) and at different developmental stages of the plant. Some barley TBPs manifested phosphatase, probably Ser/Thr or dual Ser/Thr/Tyr activity. MALDI-TOF mass spectrometry of barley TBPs identified several proteins involved in chromatin rearrangement and regulation processes, including transcription factors, serpins, protein phosphatases and protein kinases, RNA helicas- es, and DNA topoisomerase II. DOI: 10.1134/S0006297912060168 Key words: tightly bound proteins, nuclear matrix, phosphatase Eukaryotic genes are supposed to be organized into guanidine hydrochloride, etc. [6]. Serpins Spi-1, Spi-2, higher-order structures formed by discrete and topologi- and Spi-3 [7], 16 kDa protein C1D [8], some polypeptides cally independent domains organized in loops attached to with phosphatase and kinase activities, and other uniden- the nuclear matrix. The loop organization of chromatin tified polypeptides [9, 10] belong to the TBP group. Very could be important not only for compaction of the fiber, similar sets of TBPs possessing phosphatase and kinase but also for regulation of gene expression and replication. activities were found in yeast and murine myeloid ery- Polypeptides involved in specific structural organization throleukemia (MEL) cells [11, 12]. It was hypothesized and modeling of the chromatin fibers and chromosome more than fifty years ago that interactions between DNA territories are mostly insoluble nuclear matrix proteins and proteins could be covalent. This hypothesis was with strong affinity to DNA [1-5]. Functions and exis- recently proved when it was shown that 0.1-0.3% of TBPs tence of the nuclear matrix is a still-debated question, but are bound to DNA via a phosphotriester bond [13-15]. It it is evident that proteins tightly bound to DNA (TBPs) is supposed that such proteins are important for differen- are involved in chromatin folding and reorganization dur- tiation and regulation of gene activity. ing the cell cycle. TBPs are operationally defined as Despite a great deal of research, the functional sig- polypeptides that are able to form permanent or transient nificance of TBPs is not yet clear. Unfortunately, most of tight complexes with DNA, which may be stabilized by the previous studies on TBPs were performed on animal covalent bonds, and cannot be detached from DNA by tissues, and very little is known about TBPs of higher standard deproteinization procedures or by treatment plants. Characterization of plant TBPs appears to be a with strong dissociating agents such as sarcosyl, urea, challenging problem, as plant genomes are organized in a specific way and chromatin organization also possesses Abbreviations: IEF, isoelectrofocusing; MALDI TOF-TOF, some peculiarities compared to animal genomes. To fill matrix-assisted laser desorption/ionization tandem time-of-flight; this gap, we set the following goals in this work: 1) to MS, mass spectrometry; TBPs, proteins tightly bound to DNA. characterize the polypeptide spectrum of TBPs in differ- * To whom correspondence should be addressed. ent barley shoot organs; 2) to identify a set of TBPs by 679 . 680 BIELSKIENE et al. means of mass-spectrometry and protein database analy- phatase assays, PAGE, and two-dimensional IEF/PAGE sis; 3) to determine the affinity of barley TBPs to DNA; analysis. 4) to characterize phosphatase activity of barley TBPs in DNA fragment preparation. To obtain DNA frag- various shoot organs and during different plant develop- ments, the digest was incubated with proteinase K ment stages. Barley shoots were chosen as an object as a (0.5 mg/ml) and SDS (0.5%) overnight at 37°C and useful model for plant development studies. Some pre- deproteinized with chloroform. Remnants of DNA were liminary work on barley TBPs was already performed by precipitated with ethanol [20]. our group [16, 17]. It was shown that the spectrum of SDS-PAGE. TBPs (70-100 µg) were mixed with the TBPs appeared to be organ and developmental-stage spe- sample buffer, heated to 100oC for 5 min, and cooled [21]. cific and thus promising for further investigations. SDS-PAGE gels (10-12%) were run at 120 V constant volt- ages for 4 h and stained with Coomassie brilliant blue dye. Western blotting. Protein samples were fractionated MATERIALS AND METHODS by SDS-PAGE, and the gels and 0.45 µm pore size nitrocellulose filter (Bio-Rad, USA) were preincubated for Plant material. Seeds of the barley cultivar Auksiniai 15 min in transfer buffer (19 mM glycine, 25 mM Tris, 3 were obtained from the Botanical Garden of Vilnius 0.1% SDS, 20% methanol). Standard electroblotting was University (Kaire. nai, Lithuania). Etiolated shoots were performed for 3 h by applying a constant 200 mA current. grown for 3-5 days at a 24°C in darkness. Coleoptiles, first Determination of TBP phosphatase activity using leaves, and roots were dissected from shoots of Zadoks 07 MUP. 4-Methylumbelliferyl phosphate (MUP) (Boehr- (coleoptile emerged stage) and 10 (first leaf through inger Mannheim, Germany) was used as substrate at a coleoptile) developmental stages [18]. Dissected coleop- concentration of 2 mM. Hydrolysis of MUP was followed tiles, roots, and first leaf tissue were combined into one fluorimetrically (excitation and emission wavelengths were sample for each tissue at both developmental stages and 340 and 424 nm, respectively). The assay was carried out used for bulk DNA extraction. using 10 µg TBPs in 300 µl of 50 mM Tris-HCl (pH 7.0- DNA isolation. Plant tissues were frozen in liquid 9.5) or 50 mM Mes-NaOH (pH 5.5-7.0) buffer for 30 min. nitrogen and ground in a mortar up to a fine powder. Detection of TBP phosphatase activity in situ. TBPs DNA was extracted from the plant material according to were fractionated by SDS-PAGE and electroblotted onto the previously described protocol of chloroform–isoamyl nitrocellulose filters. Protein blots were incubated alcohol extraction [19] with some modifications. Cells overnight at 4°C in renaturation buffer (100 mM Hepes- (10 g) were suspended in extraction buffer (100 mM Tris- NaOH (Roth), pH 7.4, containing 0.2% CHAPS (Roth), HCl, pH 8.0, 500 mM NaCl, 50 mM EDTA, 1.25% SDS) 10 mM MgCl2, 50 mM KCl, and 1% BSA (Roth). at a cell to buffer ratio of 1 : 1.6 (v/v) and incubated at Phosphatase activity was visualized in situ by incubation 65°C for 30 min. Then extraction was performed with one of protein blots in a solution containing 50 mM Tris-HCl, volume of chloroform–isoamyl alcohol mixture (24 : pH 7.0, 0.5 % agarose, 50 µg/ml 5-bromo-4-chloro-3- 1 v/v). The final mixture was centrifuged at 4°C for 15 min indolyl phosphate (BCIP) (Sigma, USA) and 1 mg/ml at 2800g, cold ethanol (1 : 2 v/v) was added for DNA pre- nitro blue tetrazolium (NBT) (Sigma) for 2-3 h. Protein cipitation, centrifuged for 30 min at 2800g, rinsed with bands with phosphatase activity were colored blue. 70% ethanol, and air-dried. Dry DNA was dissolved in Two-dimensional gel separation (IEF/SDS-PAGE). 3 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM IEF/SDS-PAGE was performed with a Multiphor II EDTA). Digestion with RNase A (Serva, Germany) device (Amersham Biosciences, UK). Samples (20-50 µg (10 µg/ml) was performed for 3 h at room temperature. TBPs) were purified with 2-D Clean-Up Kit (Amersham) DNA was extracted with 3 ml of chloroform–isoamyl according to manufacturer’s recommendation and dis- alcohol mixture as before (24 : 1) and centrifuged at 2800g solved in sample buffer (12 g urea, 50 mg DTT, 0.13 ml (4°C). DNA was precipitated with two volumes of ethanol Pharmalit 3-10, 0.13 ml Triton X-100 and water to and 0.1 volume of 3 M sodium acetate, collected by cen- 25 ml). For the first dimension, 13 cm Immobiline Dry trifugation at 4°C for 30 min at 9000g, rinsed with 70% Strips pH 4-7 (Amersham Biosciences) were used. Dry ethanol, and air-dried. Dried DNA was dissolved in 1 ml strips were rehydrated, reduced, and alkylated according of the TE buffer and stored at 4°C. to manufacturer’s recommendations. For the second TBP sample preparation. Isolated DNA (5 mg/ml) dimension, 8-18% Excell gels (Amersham Biosciences) was diluted with 50 mM Tris-HCl buffer, pH 8.0, con- were used. SDS-PAGE buffers: anode (0.45 M Tris- taining 1 mM MgCl2 and benzonase (Merck, Germany) acetate, pH 6.6, 4 g/liter SDS, 0.05 g/liter Orange G); was added (0.1 U/µg DNA).

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