Satellite DNA binding and cellular localisation of RNA helicase P68

Natella Enukashvily1,*, Rossen Donev2,{ddagger}, Denise Sheer2 and Olga Podgornaya1

1 Cell Cultures Department, Institute of Cytology, Tikhoretsky, 4, St Petersburg, 194064, Russia
2 Human Cytogenetics Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, UK

* Author for correspondence (e-mail: natella{at}mail.ctyspb.rssi.ru)

Accepted 25 October 2004


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We purified a 68-kDa protein from the mouse nuclear matrix using ion exchange and affinity chromatography. Column fractions were tested for specific binding to mouse minor satellite DNA using a gel mobility shift assay. The protein was identified by mass spectrometry as RNA helicase P68. In fixed cells, P68 was found to shuttle in and out of SC35 domains, forming fibres and granules in a cell-cycle dependent manner. Analysis of the P68 sequence revealed a short potential coiled-coil domain that might be involved in the formation of P68 fibres. Contacts between centromeres and P68 granules were observed during all phases of the cycle but they were most prominent in mitosis. At this stage, P68 was found in both the centromeric regions and the connections between chromosomes. Direct interaction of P68/DEAD box RNA helicase with satellite DNAs in vitro has not been demonstrated for any other members of the RNA helicase family.

Key words: RNA helicase P68, Satellite DNA, Centromere


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 Introduction
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The size of genomes has increased during evolution, but the number of genes has increased only up to the level of vertebrates. In the mouse, the percentage of coding DNA in the genome is less than 5% (Waterston et al., 2002Go) and the role of tandemly organised highly repetitive non-coding sequences in the eukaryotic genome remains obscure. One class of these sequences, satellite DNA (satDNA), represents a special type of highly condensed, transcriptionally silent, constitutive heterochromatin.

Most of the satDNA is retained in so-called nuclear matrix preparations. The nuclear matrix is operationally defined as being resistant to high salt or detergents (Zbarsky et al., 1962Go; Berezney and Coffey, 1974Go). As it is associated with the protein machinery for transcription, RNA splicing and DNA replication, the nuclear matrix has been proposed to play a fundamental role in the organisation of these processes (Berezney et al., 1995Go). Although the popularity of the nuclear scaffold/matrix waxes and wanes (Hancock, 2000Go; Pederson, 2000Go), the model is presently gaining credibility (Oegema et al., 1997Go) (reviewed by Berezney, 2002Go). The nuclear matrix has undoubtedly proved its worth as a biochemical fraction enriched in specific nuclear components. A second influential concept in nuclear structure concerns the organisation of interphase chromosomes into distinct units called `territories' and this packaging in implicated in the regulation of gene expression (Cremer and Cremer, 2001Go). Constitutively active genes have been found predominantly near the periphery of chromosomal territories (Tajbakhsh et al., 2000Go). In some cases, they are on large chromatin loops extending outwards from the periphery of the chromosome territories (Volpi et al., 2000Go). The interchromosomal space is believed to contain a large number of nuclear domains that are engaged in various regulatory activities (Spector, 1993Go; de Jong et al., 1996Go; Lamond and Earnshaw, 1998Go). These include coiled or Cajal bodies, gemini of coiled bodies (gems), cleavage bodies, promyelocytic leukaemia (PML) bodies (POD or ND10 domain), interchromatin granule clusters (IGCs/speckles), and the perinucleolar compartment (PNC) (for review see Spector, 1993Go).

Although the location of the nuclear matrix in relation to chromosome territories is unclear, a hypothesis of `chromosome territories framed by the nuclear matrix' has been proposed (Podgornaya et al., 2003Go). The entire structure of the chromosome territory is preserved when most of the histones and other soluble nuclear proteins are removed, but DNA-nuclear matrix attachments are retained (Ma et al., 1999Go). These authors suggest that neither histone proteins nor disulfide bonds between nuclear matrix proteins are involved in chromatin-matrix interactions. Instead, RNA and/or ribonuclear protein interactions, together with a small subset of acidic nuclear proteins, called the `chromosome territory anchor proteins' released with the 2 M NaCl treatment, are crucial (Ma et al., 1999Go). The fact that chromosome territory architecture appears to be dependent on ongoing transcription (Haaf and Ward, 1996Go) confirms an important organisational role for either proteins or RNA.

Constitutive heterochromatin composed of satDNA has also been proposed to play a role in the organisation of chromosomal territories (Manuelidis, 1990Go). Two types of satellite DNA (satDNA) are found at the centromeric and pericentromeric regions of mouse chromosomes. One of these, minor satellite (MiSat) localises precisely to the primary constriction region (Wong and Rattner, 1988Go). In centromeric regions, MiSat monomers (120 bp) are organised in tandem repeats of approximately 300 kb (2500 copies) per chromosome. In all members of the genus Mus, which carry MiSat, in situ hybridisation has localised this DNA exclusively to the centromere of each chromosome except Y (Wong and Rattner, 1988Go; Joseph et al., 1989Go). It is difficult to draw tenable conclusions regarding the functional significance of MiSat owing to conflicting evidence (e.g. Broccoli et al., 1990Go; Vig and Richards, 1992Go). A thorough study of the proteins interacting with MiSat will undoubtedly improve our understanding of how it functions in live cells.

In primates, the centromeric DNA belongs to the alphoid satellite DNAs family ({alpha}-sat DNA). Alphoid DNA arrays are composed of a repeated 171 bp monomer (for a review, see Willard and Waye, 1987Go). In most chromosomes, {alpha}-satDNA arrays are surrounded by arrays of classical satDNA, to which human satellite 3 (HS3) belongs. The arrays of HS3 are based on variants of the ATTCCA monomeric unit (Prosser et al., 1986Go). Studies of natural and engineered rearranged chromosomes indicate that alphoid DNA is the functional centromeric DNA on most human chromosomes. The existence of stable marker chromosomes that lack alphoid DNA demonstrates that non-alphoid DNA sequences can function as centromeres (Du Sart et al., 1997Go) but the fact that all human centromeres contain some alphoid DNA suggests strongly that centromeres form most readily over alphoid DNA. HS3 has been proposed to be a part of a functional centromere (Grady et al., 1992Go).

We took the approach of screening for proteins that bind two probes containing unrelated satDNA sequences: human satellite III (HS3) and human alphoid DNA fragments (Podgornaya et al., 2000Go). A complex of three proteins (80, 70, 57 kDa, named P80, P70 and P57, respectively), with ability to bind alphoid DNA specifically, was identified from human nuclear matrix preparations by gel mobility shift assay (GMSA). None of these proteins corresponded to lamins or centromere proteins (CENP). An antibody was raised against the 70 kDa protein and immunoprecipitation analysis suggested that P70 shared a common antigenic determinant (rod domain) with intermediate filament proteins (Enukashvily et al., 2000Go). The P70 protein was also shown to be capable of interacting with HS3 and GMSA revealed no other proteins in the DNA-protein complex. DNAse I footprinting and methylation interference revealed multiple points of protection distributed throughout the HS3 fragment with a periodicity of about 10 bp, mostly inside AT islands (Podgornaya et al., 2000Go).

In mouse, the ability of the nuclear matrix protein (SAF-A/p120/hnRNP-U) to bind mouse pericentromeric satDNA (major satDNA) has been demonstrated (Lobov et al., 2000Go). Nuclear matrix proteins capable of binding MiSat that do not belong to the CENP group have not yet been described.

The aim of the current work was to find a mouse nuclear matrix protein capable of binding to MiSat. Protein purification methods and the antibodies raised previously were used to identify the protein and to trace its position in the nucleus during the cell cycle. The P68 protein identified here is a RNA helicase P68 (DEAD/H box polypeptide 5) with 98% homology to its human analogue. The protein has been localised adjacent to centromeres in a cycle-dependent manner, and shuttles between SC35 domains and the interchromatin space during the cell cycle. In chromosome preparations, the protein is found in kinetochores and interchromosome connections.


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DNA probes
A MiSat fragment (362 bp) was inserted into the pGEM7 vector using an EcoRI site. The MiSat containing plasmid (p238) was a kind gift from A. Mitchell (HGU MRC, Western General Hospital, Edinburgh, UK). Human satellite 3 fragment (HS3) (336 bp) was obtained from a larger HS3 sequence from 1q12 (1770 bp) by SauIIIA digestion and subcloned in pUC19 vector (plasmid p336) (Cooke and Hindley, 1979Go; Podgornaya et al., 2000Go). The following DNAs were also used in GMSA as competitors: 471 bp fragment of mouse major satellite cloned into pBluescript II KS+, a kind gift from B. Hamkalo (Department of Molecular Biology and Biochemistry, University of California, USA); mammalian telomeric DNA sequence (T2AG3)135 (~800 bp), excised by BamHI/BglII double digestion of plasmid pSX-neo (from R. Bayne, MRC HGU, Western General Hospital, Edinburgh); 171 bp monomer and 14-mer (~2400 bp) of human alphoid DNA both cloned into pUC19, a kind gift from A. Mitchell (Mitchell et al., 1985Go).

For GMSA, the MiSat fragment was end-labelled with [{alpha}-32P]dATP by Klenow fragment of DNA polymerase I (Sibenzyme, Russia) and purified by electrophoresis in a 1% agarose gel. For FISH and immunoFISH, Digoxigenin-11-dUTP (Roche) was incorporated into the MiSat fragment by random primed labelling or by PCR using M13 forward and reverse primers.

Preparation of nuclei and nuclear matrix
Mouse liver was homogenised in STM buffer (0.32 M sucrose, 5 mM MgCl2, 0.1 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.01% PMSF). The homogenate was loaded on a 2.1 M sucrose cushion and centrifuged for 1 hour at 4°C (80,000 g). The pellet was washed and resuspended in STM-buffer. The quality of nuclei was checked using light microscopy.

The nuclear matrix (NM) fraction was prepared essentially according to published methods (Belgrader et al., 1991Go). The pellet (NM) was resuspended in TM-5 buffer (5 mM MgCl2, 0.1 mM EDTA, 20 mM Tris-HCl, pH 7.4, 0.01% PMSF) containing 50% glycerol, and extracted immediately or stored at -20°C.

Extraction of nuclei and nuclear matrix
Nuclei were extracted with a buffer containing 10 mM Tris-HCl, pH 8.0, 0.1 mM MgCl2, 0.2 mM PMSF, 0.5 mM dithiothreitol (DTT), 5% glycerol and 0.5 M NaCl at 4°C for 1 hour, the suspension was centrifuged at 5000 g for 10 minutes. The nuclear extract (NE) fraction (supernatant) was diluted to a final NaCl concentration of 200 mM. The NM was extracted in 30 volumes of a low salt extraction buffer (25 mM Tris-HCl, pH 9.0, 10 mM EDTA, 1% 2-mercaptoethanol, 1 mM PMSF) for 12 hours at 4°C as described (Lobov et al., 2000Go). The pH was adjusted to 8.0 and NaCl added to a final concentration of 200 mM, prior to loading on a DEAE-sepharose column.

DEAE-sepharose column chromatography
The column (10 cmx1 cm) was equilibrated in a buffer solution: 15 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.12% 2-mercaptoethanol, 200 mM NaCl, 1% glycerol, 0.01% PMSF. The fractions were eluted at room temperature (step elution in the range of 0.15-1 M NaCl, in steps of 0.05 M) and stored at 4°C.

Gel mobility shift assay (GMSA)
The incubation mixture contained 1 ng [{alpha}-32P]-labelled DNA fragment, 5 µl (~5 µg of total protein) DEAE-Sepharose fraction and 0.1-5 µg of non-specific competitor DNA (Escherichia coli DNA sonicated up to average length 500 bp or linearised pUC19). The probes were incubated in the binding buffer (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1% Triton X-100, 1% 2-mercaptoethanol, 2 mM MgCl2, 5% glycerol) for 50 minutes. Electrophoresis (40 mA, 3 hours) was performed in a 4% polyacrylamide gel buffered with TAE. Gels were dried and exposed to X-ray film (CEA, Sweden). Densitometry analysis was performed using ONE-Dscan v 1.3 (Scanalytics, CSP Inc, USA) software.

Affinity purification of P68 and mass spectrometry identification
Either the nuclear matrix extract or the DEAE-fraction that bound MiSat most intensely (an `active fraction') was subjected to further purification by affinity chromatography. Biotinylated MiSat was immobilised on streptavidin-coated magnetic beads according to the manufacturer's protocol (Roche). The nuclear matrix extract was incubated with the beads in GMSA binding buffer in presence of competitor DNA (either E. coli DNA, p238 or pUC19) for 1 hour at 4°C in conditions described (Enukashvily et al., 2000Go). The beads were then washed three times for 5 minutes in GMSA binding buffer containing 0.25 M NaCl and the same amount of competitor DNA as used for the incubation. Finally, the beads were washed in binding buffer containing 0.5 M NaCl and boiled in Laemmli sample buffer. The last two fractions were subjected to SDS-PAGE and immunoblotting. P68 was cut out of a Coomassie-stained gel and processed further for mass spectrometry as described previously (Pappin et al., 1993Go). Mass spectrometry was performed at Cancer Research UK, London Research Institute facilities in three attempts, each of them giving a protein identity with 97% probability.

SDS-PAGE and immunoblotting
SDS-PAGE (10% and 12%), semi-dry protein transfer onto nitrocellulose and immunoblotting as well as silver and Coomassie Brilliant Blue staining were performed according to standard protocols. The following antibodies were used for immunoblotting: mAbs against lamin B and lamins A/C (Vector Labs, USA), pAb against splicing factor SC35 (Sigma), pAb R-288 (rabbit serum against 14 kDa C-end fragment of p80-coilin) (Andrade et al., 1993Go), pAb against CENP-B (Earnshaw et al., 1989Go) and pAb against p70 (Enukashvily et al., 2000Go). We also used IFA antibody, which recognises the epitope YRKLLEGEE within the rod domains of intermediate filament (IF) proteins (Riemer et al., 1991Go; Pruss et al., 1981Go).

Cells and synchronisation
Mouse L929 and human HeLa cells were grown in RPMI medium supplemented with 10% foetal calf serum (Gibco) up to 60% confluence, washed with phosphate-buffered saline (PBS) and fixed as described below. In synchronisation experiments, cells were blocked in G1 phase by serum deprivation for 48 hours (Table 1, I). The block was released by adding medium containing fresh serum. To enrich S-phase cells, L929 cells were harvested in 12-13 hours after serum replenishment (Table 1, II). To arrest cells in G2/M phase, they were collected 14 hours after serum addition (Table 1, III). The percentage of cells in G1, S and G2/M phase was determined by FACS analysis of samples treated in parallel according to the standard protocol.


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Table 1. FACS analysis of L929 cell synchronisation

 

In the immunofluorescence and immunoFISH experiments, the time within S phase was determined by FISH with a MiSat probe. In all cells where replication timing was assessed, centromeres have been shown to replicate at the very end of S phase (e.g. Pudenko et al., 1997Go; Hultdin et al., 2001Go). At the beginning of S phase, 4',6-diamidino-2-phenylindole (DAPI)-stained chromocentres were reported to be the same size as in G1, whereas at the end of S phase, when the replication of centromeres proceeded, they became very small and numerous. Therefore, we assumed that an S-phase cell with non-replicated centromeres and chromocentres of the same size and number as in G1 should be at the early stage of S phase. A cell with replicated centromeres and small and numerous chromocentres was considered as passing through the final stage of S phase.

The method used for cell synchronisation allowed us to obtain an approximately equal number of cells in G1 and G2 phases 14 hours after serum addition (Table 1). The percentage of cells in G2/M phase was additionally monitored by a method of integral brightness measurement of nuclei counterstained with DAPI. The densitometry of the images taken by CCD camera was performed using the ImageTest software (Institute of Cytology, St Petersburg). In this method, cells in G2/M phase should stain with intercalating dyes twice as brightly as G1/G0 cells. The measurement of total nucleus DAPI fluorescence brightness (calculated as a sum of all image pixels in one nucleus) is a measurement of DAPI incorporation. Hence, on the same slide, this parameter will be twice as high for G2/M cells as for G1/G0 cells. The images were used to analyse centromeres and P68 distribution in G2 only after this integral brightness measurement.

Chromosome spreads and prematurely condensed chromosome preparation
To obtain chromosome spreads, colcemide was added into the cell medium to a final concentration of 50 ng/ml for 1 hour. Cells were hypotonically treated with 0.075 M KCl, resuspended in methanol:acetic acid (3:1), spread onto glass slides and air-dried. Alternatively, cells were spread onto glass after treatment with KCl and fixed with 1% paraformaldehyde.

To obtain prematurely condensed chromosome (PCC) preparations, mouse zygotes (first mitotic division, 26 hours after the administration of human chorionic gonadotropin) washed out of oviducts of superovulated fertilised mice were treated with okadaic acid (final concentration 5 µM) as described previously (Dyban et al., 1993Go). Briefly, the zygotes without zona pellucida were transferred to drops of M16 medium (Sigma) with okadaic acid and incubated under paraffin oil at 37°C, 7.5% CO2 for 45 minutes. Treatment with okadaic acid (a specific inhibitor of phosphoprotein phosphatases 1 and 2A) induces nuclear envelope fragmentation and premature condensation of interphase chromosomes in pronuclei as well as in secondary polar body nuclei (Cohen et al., 1990Go; Dyban et al., 1993Go). Subsequent treatment with series of fixatives containing methanol and acetic acid in different proportions (75% glacial acetic acid:methanol, 1:1; glacial acetic acid:methanol, 1:1; methanol:glacial acetic acid, 4:1) enables PCC preparations that are free of cytoplasmic contaminations, nuclear envelope or nucleoplasmic contaminations to be obtained. These preparations have chromosomes with different degrees of condensation attached to the nucleolus. The maximal number of chromosomes in one PCC rosette does not exceed 20 as the zygotes were obtained prior to the male and female pronuclei fusion. The degree of chromosome condensation depends on the cell cycle stage (Dyban et al., 1993Go; Dozortsev et al., 2000Go). If the fixation step is preceded by non-ionic detergent treatment, the proteins within the pronucleolar body are removed and an interchromosome connection becomes clearly visible. On the slides used in the present work, the PCC preparations were treated with 1% Tween-20 in PBS for 15 minutes at room temperature and fixed. They were classified as G2 chromosomes according to morphological criteria (Dyban et al., 1993Go).

Indirect immunocytochemistry
L929 cells were fixed in KCM buffer (135 mM KCl, 20 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100, 10 mM Tris-HCl pH 8.0) containing 2% PFA for 30 minutes at room temperature. Slides with fixed cells were washed in KCM, blocked with 10% bovine serum albumin (BSA) and incubated for 1 hour with antibodies against P68. FITC-conjugated anti-guinea pig antibodies (VectorLabs) at 1:75 dilution were used as secondary antibodies. Finally, cells were counterstained with DAPI (0.5 ng/µl for 4 minutes) and mounted in Vectashield mounting medium (VectorLabs, USA). For double immunofluorescence, P68 antibody was detected by FITC-conjugated anti-guinea pig antibody. After fixation in KCM containing 2% paraformaldehyde (10 minutes, at 4°C), cells were washed and incubated with antibody either against SC35 (Sigma) (1:200), coilin (1:100) or CENP-B (1:100) for 1 hour. The secondary antibodies were conjugated to Texas Red.

ImmunoFISH
MiSat, HS3 (Podgornaya et al., 2000Go), {alpha}-satDNA (Enukashvily et al., 2000Go), labelled with digoxigenin-11-dUTP (Roche) were used as FISH probes. L929 cells were treated for immunostaining as described above. To proceed with FISH, cells were incubated in 2xSC at 37°C for 10 minutes and denatured in 70% formamide/2xSC at 74°C for 7 minutes. The denatured hybridisation mix (2 mg/ml of labelled DNA, 2xSC, 50% deionised formamide, 10% dextran sulphate, 20 mg/ml E. coli DNA) was applied to the slides. Hybridisation was performed at 41°C overnight. Slides were then washed in 50% formamide/4xSC (twice for 5 minutes each), 50% formamide/2xSC (four times for 5 minutes each) at hybridisation temperature, once in 0.5xSC at room temperature and blocked with BSA. The hybridisation sites of MiSat probe were detected by anti-digoxigenin antibody conjugated with Rhodamine (Roche). Cells were counterstained with DAPI and mounted in antifade medium.

Images were recorded with a Zeiss Axioplan epifluorescence microscope equipped with a CCD camera (Sony) controlled by KS100 software (Carl Zeiss, Germany). The images were pseudocoloured red, green or blue, merged using Corel PhotopaintTM or Adobe PhotoshopTM software and prepared for publication with CorelDrawTM.

Sequence analysis of RNA helicase P68
Mouse and human RNA helicases P68 (NCBI protein database accession numbers: XP_122265 and XP_008344) were aligned to rod domains of human and mouse lamins B2 (XP_008344 and P21619) and to human, mouse and rat matrins 3 (P_43243, AAH_29070 and P_43244) using CLUSTALW software (Higgins et al., 1994Go). The same software was used to align RNA helicase P68 with IFA epitope. Secondary structure prediction of P68 from primary sequence was carried out with PREDATOR software (ftp://ftp.ebi.ac.uk; directory: /pub/software/dos/predator). The probability of coiled-coil formation was estimated by COILS software (http://www.ch.embnet.org/software/COILS_form.html). The basic principle of COILS is to search within a protein sequence for the heptad repeat pattern, which is a signature of the coiled-coil motif (Lupas et al., 1991Go). The COILS program scans the analysed sequence for the heptad pattern in three different modes: searching for double (14 amino acids), triple (21 aa) and quadruple (28 aa) heptad units. It is assumed that residues with probabilities >50% might be a part of a coiled-coil segment especially if they are adjacent to the regions with probabilities >80%.


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RNA helicase P68 is a MiSatDNA binding protein
NE and NM extracts, both fractionated by DEAE-chromatography, were tested by GMSA for binding to MiSat probe (Fig. 1). Both the NM and the NE extracts interacted with the MiSat fragment. The binding activity was observed in NM and NE fractions eluted in 0.3-0.4M NaCl. However, under the same experimental conditions, the NM fractions eluted by 0.3-0.4M NaCl bound 79% of labelled probe, whereas the NE bound only 28% of MiSat according to densitometry analysis. Hence, the major fraction of MiSat binding activity was retained in the nuclear matrix. The NM fraction eluted by 0.3 NaCl ('the active fraction') was chosen for further work. Several complexes were revealed in autoradiographs when this fraction was added into the incubation mix. Two of these were stable in the presence of up to 75-fold excess of competitor DNA. However, the complex that migrated more slowly disappeared at 150-fold excess while the other one was still detectable (Fig. 1). Thus, the upper complex was less stable. Both complexes dissociated when 50-fold excess of unlabelled MiSat but not pUC19 was added to the mix (Fig. 2). These data therefore confirmed the specificity of the NM fraction binding to the MiSat probe. The major satellite DNA (MaSat) and HS3 competed for binding to some extent, however, telomere repeat did not show any affinity to the protein fraction (Fig. 2B). Plasmid with alphoid DNA (human centromeric DNA) monomer insertion (170 bp) was a better competitor than MaSat and plasmid with inserted 14-mer alphoid DNA competed for binding even more effectively than MiSat itself (Fig. 2B). Centromeric DNAs were thus the strongest competitors, whereas pericentromeric satDNAs competed for binding to a much lesser extent.



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Fig. 1. Gel mobility shift assay (GMSA) of the nuclear and nuclear matrix extracts from mouse liver cells. Nuclear extract (NE, lanes 2-8) and nuclear matrix extract (NM, lanes 9-15) DEAE fractions (5 µg of total protein) were eluted from a DEAE-column by 0.2 M (lanes 2, 3, 9, 10), 0.3 M (lanes 4-6, 11-13) and 0.4 M (lanes 7, 8, 14, 15) NaCl. 1 ng of labelled MiSat fragment was added to the incubation mix. Each probe contained either 50-, 75- or 150-fold weight excess of E. coli DNA as indicated. The GMSA of the mock probe (without proteins added) is shown in lane 1. The fraction of the nuclear matrix eluted by 0.3 M NaCl was chosen for further work. The arrowheads point to the border between two sets of lanes (NE and NM).

 


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Fig. 2. GMSA of the 0.3 M NaCl mouse liver nuclear matrix fraction in the presence of different competitors. (A) E. coli DNA (50, 75, 150 weight excess, lanes 2, 3, 4, respectively), HS3 (lane 5), pUC19 (lane 6) and minor satellite (MiSat) (lane 7). HS3, pUC19 and MiSat were loaded in 50-fold weight excess. The mock probe GMSA is shown in lane 1. (B) The GMSA of the same fraction as in A in the presence of the competitors: dIdC, 100-fold weight excess to the labelled miSat probe (lane 2), pUC19 (lanes 3-5), MiSat (lanes 6-8), major satellite (MaSat) (lanes 9-11), telomere repeats (tel) (lanes 12-14), pUC 19 containing alphoid DNA monomer ({alpha}) (lanes 15-17), pUC19 with alphoid DNA 14-mer insertion ({alpha}x14) (lanes 18, 19). All the competitors (except dIdC) were loaded in 50-(lanes 3, 6, 9, 12, 15, 18), 75-(lanes 4, 7, 10, 13, 16, 19) and 150-fold (lanes 5, 8, 11, 14, 17) weight excess. The mock probe GMSA is shown in lane 1.

 

Affinity chromatography was performed using streptavidin-coated beads with immobilised biotinylated MiSat (Fig. 3). After a series of washes, the NM extract proteins bound to satDNA were eluted by 0.5M NaCl (Fig. 3A, lane 3) or the beads were boiled in SDS-PAGE sample buffer (Fig. 3A, lane 4). A protein of 68 kDa (P68) was the major protein in both these fractions. Most of the protein was removed from beads only after boiling in SDS buffer (Fig. 3A, lane 4), suggesting a high affinity of binding. The specificity of binding was checked by incubation of the NM extract with different competitors during affinity chromatography: pUC19 or p238. P68 was not detectable in fractions in the latter case (Fig. 3B, lane 3). Lamin B was found to be a contaminant of p68 by subjecting the NM extract fraction (Fig. 3B, lanes 3, 4) to immunoblotting with the antibody against lamin B (data not shown).



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Fig. 3. Affinity purification of p68 on streptavidin-coated magnetic beads with biotinylated MiSat fragment. The fractions were subjected to 12% (A) or 10% (B) SDS-PAGE and stained with Coomassie Blue. (A) Lane 2, NM proteins; lane 3, NM proteins washed from the beads by 0.5 M NaCl in GMSA buffer; lane 4, proteins washed from the beads by boiling in SDS-PAGE sample buffer. (B) Influence of competitor DNA (MiSat, lane 3; pUC19, lane 4) on the eluate composition. The probes in lane 4 contained 50-fold excess of pUC19, in lane 3, 10-fold weight excess of MiSat containing p238 as competitor DNA when loaded into affinity column. The nuclear matrix extract loaded is shown in lane 2. P68 and lamin B (L) bands are indicated. The binding activity of fractions in GMSA is indicated at the top. Size markers are shown in lane 1 of panels A and B.

 

Lamin B has been shown to bind satDNA (Lobov et al., 2001Go). Immunoblots with anti-lamin antibodies were performed in order to check if contamination by lamins was responsible for the MiSat binding activity. Lamins A/C and B were present in the NM extract but not in the DEAE fraction (Fig. 4). Therefore, lamin B from the NM extract could bind MiSat in affinity chromatography but it was not responsible for the MiSat binding to P68 demonstrated by GMSA (Fig. 1).



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Fig. 4. Western blotting of the GMSA nuclear matrix extract and DEAE-purified fractions. Coomassie Blue staining (1-3) and immunoblot (4-9) of the nuclear matrix extract (lanes 2, 4, 6, 8) and 0.3 M NaCl DEAE fractions (lanes 3, 5, 7, 9) detected with antibodies against P68, lamins A/C or lamin B as indicated.

 

Alphoid DNA was the strongest competitor against MiSat in GMSA (Fig. 2). The molecular weight of mouse P68 was very similar to that of human alphoid DNA binding protein described as P70, against which an antibody had previously been raised (Enukashvily et al., 2000Go). Therefore, we checked whether the serum raised was suitable for the mouse proteins. The serum added to the probe for retardation caused hypershift of the complexes (data not shown) and in the DEAE fraction it revealed a protein of 68 kDa (Fig. 4, lane 5). Thus mouse P68 and human P70 share high homology domains and the antibody against P70 can be used to trace P68 in mouse cells.

After additional purification by immunoprecipitation, P68 was identified as the RNA helicase P68 (DEAD/H box polypeptide 5) by mass spectrometry. The mouse RNA helicase P68 shares high homology, 98% by amino acid sequence, with the human form (Lemaire and Heinlein, 1993Go). As a result of the high homology, the antibody raised against human p70 is able to recognize mouse P68. Below we use the designation `the antibody against P68' for the antibody raised against P70 (Enukashvily et al., 2000Go) and `P68' for the protein recognised by this antibody.

Sequence analysis of RNA helicase P68
The nuclear matrix has been visualised by electron microscopy as a network of 7-10 nm filaments with an appearance similar to that of the cytoplasmic intermediate filaments (Traub, 1995Go). Cross-reaction of P68 with the IFA antibody was shown in our previous work (Enukashvily et al., 2000Go). A computer search for the IFA epitope (see Materials and Methods) within P68 RNA helicase revealed the region of 518-529 amino acid (aa) residues as a region of the highest homology with intermediate filaments. The homology was not perfect, but sufficient to make the protein recognizable by the IFA antibody.

Another common feature of intermediate filaments is the presence of coiled-coil domains that are essential for protein dimerisation and higher order intermediate filament protein organisation (e.g. Strelkov et al., 2002Go). Analysis of the secondary structure of P68 predicted from the primary sequence revealed that region 301-370 aa is a potential coiled-coil domain (Fig. 5A). The probability did not exceed 62% in quadruple (the strictest) COILS mode, although in double heptad mode the region 349-369 aa revealed 95% probability (Fig. 5A). Taking into consideration the fragment length, as recommended by the COILS developers, we predicted that the 301-370 aa region might be involved in protein-protein interaction based on coiled-coil structure, with 349-369 aa being the most probable site of heptad repeat pattern formation (Fig. 5A,B). The rod domain of intermediate filaments proteins has an {alpha}-helical structure (Geisler et al., 1992Go). Thus, the predicted {alpha}-helical structure of P68, especially in the region 349-369 aa (Fig. 5B) strengthens the suggestion of a short coiled-coil domain within P68.



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Fig. 5. Computer analysis of the amino acid sequence of P68. (A) Results of potential coiled-coil domain search using COIL software. x-axis shows the amino acid residues starting from N-terminus, y-axis, the coiled-coil formation probability, P. (B) Computer alignment of human/mouse P68 (h/m P68) with mouse (mLB) and human (hLB) lamins B2 rod domain fragments. Potential coiled coils shown in A are boxed in yellow (P=0.62) and in orange rectangles (P>0.95). The green rectangle is coil 1A, the blue rectangle is coil 1B. The line below the alignment shows the similarity score: *, amino acids in that column are identical;:, conserved substitutions;., semi-conserved substitutions. The lines labelled p68 PrSS and LB PrSS represent prediction of the secondary structure of P68 and lamin B2, respectively, from its primary sequence. H, a region of {alpha}-helical structure; E, extended sheet; _ flexible coil structure.

 

Lamins are the most well documented intermediate filament rod-domain-containing nuclear proteins. We therefore searched for regions of homology with these proteins. The best results were obtained for a P68 fragment DYIHINIGALELSANHNILQIV, 297-319 aa, which was then used as a reference point for computer alignment. The aligned lamin B2 sequence (HYIDRVRALELENDRLLLRIS) resides in the most conservative part of the coil 1A structure (Strelkov et al., 2002Go) of human and mouse lamin B2 (Fig. 5B). Maximum similarity was observed between coil 1A of the lamin B2 rod domain and the region 285-337 aa of P68, which partially overlaps the region (301-370 aa) where heptad pattern formation was predicted (Fig. 5B). Similarity was also found between coil 1B of the lamin B2 rod domain and a region of P68 that encompasses the potential coiled-coil region (349-369 aa). Although the P68 protein may not have a fully functional rod domain of intermediate filaments, its potential coiled coil region with homology to lamins suggests an involvement in nuclear filament formation.

Matrins have been claimed to be elements of the nuclear matrix. We tried to align p68 to matrin 3 of human, mouse and rat. All the regions of homology (4-12 aa) were found within the ATPase domain region (567-776 aa) of the matrins aligned to the region 420-577 aa of mouse p68. This might be explained by the fact that both matrins and p68 need to utilise ATP to perform their functions.

Distribution of P68 helicase in the nucleus
In human HeLa cells, we analysed the spatial relationship between P68 and alphoid DNA or HS3 as the protein was shown to bind specifically to these DNAs in vitro. P68 was partly adjacent to centromeres in G0/G1 cells (Fig. 6A). Perfect correspondence between P68 granules and centromeres was rarely observed. The main P68 signal did not correspond to HS3 domains (Fig. 6B).



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Fig. 6. ImmunoFISH of HeLa cells fixed with 2% PFA for 30 minutes. Antibodies against P68 (green) and alphoid DNA (A) and HS3 (B) probes (in red) were used to probe HeLa cells. (C) Double immunofluorescence staining of HeLa cells with SC35 antibody (red) and the antibody against P68 (green). Bar, 10 µm.

 

In mice, we analysed the spatial relationship between P68 and chromocentres. `Chromocentre' is a term suggested for constitutive heterochromatin domains brightly stained with intercalating dyes (e.g. DAPI or Hoechst). The number of chromocentres is cell cycle dependent and tissue specific (Pudenko et al., 1997Go; Cerda et al., 1999Go).

During the entire interphase, P68 did not invade but framed chromocentres stained with DAPI (Fig. 7). In synchronised L929 cells, P68 forms granules (0.2-1 µm) and fibres in G1 and G2 (Fig. 7A,D) but not in early S phase (Fig. 7B). In early S phase, P68 is distributed diffusely with no detectable fibres or granules (Fig. 7B). Later, at the end of S phase (Fig. 7C), P68 again forms fibres and granules.



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Fig. 7. Immunofluorescent staining of synchronised mouse L929 cells with antibody against P68. Cells were stained with anti-P68 (green) and counterstained with DAPI (red pseudocolour) to reveal the location of p68 with regard to chromocentres, and were captured in G1 (A), early S (B), late S (C), G2 (D). The first column shows merged images. Bar, 10 µm.

 

Nuclear domains of various function and composition have been described. We tried to determine whether the P68 granules correspond to any known nuclear domain. No correspondence was found between P68 granules and coiled bodies revealed by anti-coilin antibody (data not shown). However in G1, P68 granules were detected in SC35 speckles (a storage depot for many splicing factors and other nuclear proteins) as shown by double immunofluorescence staining with SC35 antibody of human (Fig. 6C) and mouse cells (Fig. 8A). No colocalisation was observed between SC35 and P68 fibres. In G2, most P68 granules and fibres did not coincide with SC35 domains (Fig. 8B). Hence, during the cell cycle, the protein shuttles between different nuclear domains. The position of centromeres with respect to SC35 domains was studied by double immunofluorescence (Fig. 9). Partial contacts between centromeres and SC35 domains were observed in G1.



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Fig. 8. Double immunofluorescence staining of mouse L929 cells with antibodies against P68 and SC35. P68 (green) and SC35 (red) staining of cell nuclei at G1 (A) and G2 (B) with merged images in the first column. Bar, 10 µm.

 


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Fig. 9. Double immunofluorescence of SC35 and centromeric protein in L929 cells. Cells in G1 show the spatial proximity of some centromeres stained with antibody against CENP-B (III, red) to SC35 domains (II, green). Cells were counterstained with DAPI (IV, blue). Merged image (I) Bar, 10 µm.

 

As our data showed that RNA helicase P68 interacts with mouse centromeric DNA in vitro (Figs 1, 2), our next aim was to examine distribution of this protein in nuclei in relation to centromeres. In G1, colocalisation of centromeres to P68 fibres was observed rarely (Fig. 10A). However, most of the centromeres (76±8% per cell) revealed by FISH were adjacent to the P68 granules. At the beginning of S phase, centromeres were positioned exactly at the borders between chromocentres and diffusely distributed P68 (Fig. 10B). Later, at the end of S phase, contact between replicated centromeres and the fibrogranular network of P68 was more obvious (Fig. 10C). In prophase when condensation of chromosomes has begun, the fibrous material stained with the anti-P68 antibody became clearly visible and centromeres were observed associated with the fibres (Fig. 10D). In these cells, the MiSat probe hybridised not only with centromeric regions but also with the thread between them.



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Fig. 10. ImmunoFISH of L929 mouse cells at various cell stages. Cells at G1 (A), early S (B), late S (C), late G2-early prophase (D) were used for colocalisation studies with P68 and FISH with a MiSat probe. Column I, merged image (P68 in green, centromeric proteins in red); II, staining with P68 antibody; III, FISH with MiSat probe; IV, DAPI staining (light blue). Bar, 10 µm.

 

Distribution of P68 helicase in mitosis
Perfect correspondence of P68 to DAPI-stained chromocentres had been observed in ~20% of 3T3 mouse monolayer cells (Enukashvily and Podgornaya, 2001Go). In a synchronised L929 culture, at late S phase, some of the chromocentres were stained with P68 antibody (Fig. 7C, Fig. 10C). The P68 signals abutted the centromeres and often overlapped them. We observed marked correspondence of P68 with chromocentres and centromeres in late S phase just before condensation of chromosomes. In early prophase the P68 signal corresponded mostly to centromeres and the connection between centromeres was visible when MiSat was used as FISH probe (Fig. 10D).

Physical interchromosome connections containing satDNA, RNA and proteins have been observed previously (Hoskins, 1968Go; Bennet et al., 1983; Maniotis et al., 1997Go; Dozortsev et al., 2000Go; Saifitdinova et al., 2001Go). Helicase P68 appears to be a protein constituent of the connecting thread as it was observed in metaphase spreads (Fig. 11A,B). Prematurely condensed G2 chromosomes isolated from mouse pronuclei allowed us to clarify the relationship between P68 helicase and the connecting thread (Fig. 11C). During preparation of the metaphase plate, none of the 20 chromosomes was lost, although their integrity was slightly altered. The thread between chromosomes was visible by DAPI staining, indicating that it contained DNA, and was delineated by the P68 helicase signal. In chromosomes, the strongest P68 signal was found in their primary constriction regions and weaker staining in chromosome arms (Fig. 11C).



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Fig. 11. Immunofluorescence of L929 mitotic chromosome spreads. Cells spreads were fixed with methanol:acetic acid (A), with paraformaldehyde (B) and prematurely condensed G2 chromosome preparation (C). P68 is shown in green. Chromosomes were counterstained with DAPI and pseudocoloured in red. Centromeres stained with an antibody against P68 are indicated with white arrowheads; the interchromosome connection is indicated with black arrowheads. Bar, 20 µm.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The DEAD box protein family of RNA helicases includes over 40 proteins from a wide range of organisms. These proteins share a group of conserved motifs including the sequence Asp-Glu-Ala-Asp (D-E-A-D) that provides their name (Linder et al., 1989Go; Fuller-Pace, 1994Go). DEAD box proteins are implicated in diverse cellular functions including splicing, ribosome assembly, translation initiation, spermatogenesis, mRNA stability, embryogenesis, cell growth and division. Some members of the DEAD box and related families (i.e. DEXD-box) may function on RNA-RNA, RNA-DNA or DNA-DNA duplexes (Linder, 2000Go). P68 is one of the prototype DEAD box proteins, originally isolated through a cross-reaction with a monoclonal antibody against SV40 large T antigen (Lane and Hoeffer, 1980Go). The purified protein P68 has been shown to exhibit RNA-dependent ATPase activity and functions as an RNA helicase in vitro (Hirling et al., 1989Go; Iggo and Lane, 1989Go). Human RNA helicase P68 has been detected in the nuclear matrix (Akileswaran et al., 2001Go).

Interaction between P68 and centromeric satellite DNA in vitro
The biological functions of P68 remain to be established. This is a nuclear protein with an RNA binding motif YIHRIGRTAR and two C-terminal RGG boxes. Although the RGG motif is shorter than in other proteins, it may be involved in the interaction of P68 with RNA (Burd and Dreyfuss, 1994Go). Furthermore, P68 RNA helicase appears to be one of the main satDNA binding proteins in vitro (Figs 1, 2, 3, 4) (Podgornaya et al., 2000Go). Although this is the first RNA helicase to be associated with centromeric satDNA, the putative transcriptional regulator ATRX, which is a member of the SNF2 family of helicase/ATPase proteins, has also been found at pericentromeric heterochromatin (McDowell et al., 1999Go). It has been suggested that proteins of the helicase/ATPase superfamily may be incorporated into multicomponent complexes that utilize energy from ATP hydrolysis to remodel chromatin and thus, to regulate protein/DNA interactions (Kingston et al., 1996Go). The conditions required to solubilise ATRX from cellular extracts suggest its tight association with chromatin. Thus, it was proposed that ATRX might bind DNA (Villard et al., 1997Go). However, the association of ATRX and DNA-binding mHP1a in a two-hybrid assay (Le Douarin et al., 1996Go) and colocalisation of ATRX with antibody against HP1b shows that the proteins directly interact and ATRX association with DNA could be via HP1. As for P68, the protein was the major component of the affinity-purified fraction. Thus, P68 interacts directly with MiSat DNA.

Homology with intermediate filament proteins
As RNA helicase P68 is a nuclear matrix component, we examined its homology and structural similarity with intermediate filament proteins. Homology with lamin B2 was found to span 150 amino acids of P68 (Fig. 5). Although rod-domain like motifs of ~50 amino acids are present in P68 RNA helicase, this does not mean that the protein belongs to intermediate filament family. The spatial conformation of this helicase is different from that of intermediate filament proteins (Tanner and Linder, 2001Go). A short potential coiled-coil domain has, however, also been described in the helicase ATRX (McDowell et al., 1999Go). We thus suggest that both the homology with lamin B2 and the flexible spatial conformation of helicases might provide the molecular basis for protein involvement in filaments and association with the nuclear matrix in vitro.

Intranuclear localisation of P68
It has been shown that human P68 undergoes changes in nuclear localisation during the cell cycle (Iggo and Lane, 1989Go; Iggo et al., 1991Go). A nucleolar protein, fibrillarin, has been found to interact with P68 (Nicol et al., 2000Go). However, immunofluorescence studies revealed that in interphase cells, fibrillarin is predominantly nucleolar, whereas P68 shows a diffuse granular nuclear staining but is largely excluded from the nucleoli. P68 transiently enters pre-nucleolar bodies during telophase (Iggo et al., 1991Go; Nicol et al., 2000Go). We have not observed nucleolar staining (Fig. 7), probably because telophase is short and therefore is rarely seen in synchronisation experiments. The location of P68 in other cell cycle stages is consistent with published data. During interphase, P68 is found in the nucleoplasm and excluded from the nucleoli. The granular pattern of P68 distribution is well documented (Lane and Hoeffer, 1980Go; Lamm et al., 1996Go; Nicol et al., 2000Go).

P72, a human nuclear DEAD box protein and a very close homologue of P68, has recently been shown to form heterodimers with P68 in vivo (Ogilvie et al., 2003Go). P72 has a predominantly granular nucleoplasmic staining pattern, excluding nucleoli, like P68 (Lamm et al., 1996Go). As several DEAD box proteins are involved in pre-mRNA splicing, these authors tested whether P72 localises to splicing snRNP-enriched coiled bodies. No overlapping of P68 or P72 signals with coiled bodies was found in that study or in the current work

During G1/G0, we observed RNA helicase P68 within SC35 domains (Fig. 6C, Fig. 8). A biochemical procedure has been reported for the isolation of SC35 domains/speckles/interchromatin granules clusters (IGC) (Mintz et al., 1999Go). The first steps of this procedure are identical to that for nuclear matrix preparations (Zbarsky et al., 1962Go; Berezney et al., 1995Go). Therefore, IGC proteins were assumed to be components of the nuclear matrix and indeed, have been found in the nuclear matrix (Turner and Franchi, 1987Go; Jagatheesan et al., 1999Go; Martelli et al., 1999Go).

In G1 cells, RNA helicase P68 is often found adjacent to centromeres (Fig. 10). It has been demonstrated that centromeres are usually localised on the surface but not in the interior of the chromosome territory (Everett et al., 1999Go; Chevret et al., 2000Go). A cell cycle dependent, proteasome-mediated association of centromeres with PML (ND10) domains has been shown (Everett et al., 1999Go). Hence, centromeres are dynamically associated with transcription-related nuclear domains. This observation may provide a clue to understanding how the structure of a chromosome territory is organised. At most cell cycle stages, we observed P68 granules or fibres framing regions brightly stained with DAPI. Thus, if P68 RNA helicase is involved in transcription, its position is in accordance with the suggestion that the structure of chromosome territories is, at least in part, driven by transcription (Haaf and Ward, 1996Go; Volpi et al., 2000Go; Mahy et al., 2002Go).

Many nuclear matrix proteins are ribonucleoproteins that function in mRNA processing (Mattern et al., 1996Go; Tan et al., 2000Go; Lobov et al., 2000Go; Donev et al., 2003Go) and P68 helicase could be one of these proteins. On the other hand, as a MiSat binding protein it could be involved in the anchoring of centromeres to the nuclear matrix.

In early S phase, P68 distributes diffusely but later, at the end of S phase, beginning of G2, P68 is assembled again into fibres and granules (Figs 7, 10). P68 granules, which appear again at G2, do not correspond to speckles/SC35 domains (Fig. 8B). The change in P68 pattern from G1 through S to G2 is likely to be a result of function specific for the S phase only, such as DNA-replication. Centromere-P68 association becomes prominent at late S/G2 when replication is finished (Fig. 10D). In G2, centromeres colocalize mainly with the P68 fibres (Figs 7, 10). However, the granules at this stage might be too small to resolve by microscopic visualisation.

Recently, stress-induced transcription of HS3 repeats of 9q12 was observed (Jolly et al., 2004Go). The HS3 RNA remained associated with 9q12 region even throughout mitosis. RNA-binding proteins such as RNA-dependent RNA polymerase (Volpe et al., 2002Go) or NAP57/dyskerin, the orthologue of yeast Cbf5p (Jiang et al., 1993Go), are known to be satDNA or centromeric binding proteins. The presence of RNA helicases in heterochromatic regions could thus be expected. In G2 P68 starts moving to centromeres and the thread to reside there during mitosis, this is probably one of the steps in assembling the mitotic plate.

Localisation of P68 in mitosis
On mitotic chromosomes, P68 was detected in and around the primary constriction region and in interchromosome connections (Fig. 11). Physical connections between the chromosomes have been previously identified (Bennet et al., 1983; Maniotis et al., 1997Go), and shown to be an artefact of neither colchicine nor hypotonic treatment (Takayama, 1975Go; Takayama, 1976Go; Chiarelli et al., 1977Go). In mouse pronuclei, interchromosomal connections consist of satDNA (Dozortsev et al., 2000Go). Similar results have been obtained for the satDNA fragment FCP in the chaffinch (Fringilla coelebs PstI) (Saifitdinova et al., 2001Go). The authors suggest a role for DNA-protein interactions in thread formation based on their experimental findings.

It has been demonstrated that the interchromosome connection in mammalian cells can be completely destroyed with DNase (Maniotis et al., 1997Go) but when treated with RNase or pepsin it becomes more rigid: on relaxation it does not return to its original length and fibres form a coiled configuration indicating loss of elasticity (Hoskins, 1968Go). Electron microscopy revealed four groups of fibres attached to the centromeres of mouse and human chromosomes. Each of these four groups appears to be about 600 nm thick and to be made up of smaller fibres that are bound into a fascicle by a smaller very dense spiralling fibre less than 100 nm thick. Only the dense spiralling fibre is small enough to be a DNA strand. The larger fibres are presumably protein. Thus, the thread is composed of DNA fibres of 100 nm wrapped around the proteinaceous fibres of 600 nm (Hoskins, 1968Go).

In all the papers cited, conventional mitotic plates or spreads were used to reveal interchromosome connecting threads in vivo and in vitro. We observed that the interchromosome connection in both spreads and PCC preparations from cultured and germ cells contains RNA helicase P68 (Fig. 11). This is the first proteinaceous component to be found in the connecting thread. Intermediate filament rod-domain-like motifs within P68, as well as the P68 specific binding to satDNA are the features that make this protein a suitable candidate for establishing centromere connections in mitosis.


    Acknowledgments
 
This work was supported by grants from The Wellcome Trust (UK), HUGO DOE (USA), The Royal Society (UK), Russian Foundation for Basic Research, and in part by Cancer Research UK. We are thankful to R. Berezney for his help at the beginning of this work. This work could not be finished without A. Mitchell and R. Dey. We thank A. P. Dyban and E. M. Tumanishvily for their great scientific and technical help in work with PCC preparations. We are also grateful to I. S. Kuznetsova and A. S. Kukalev for their assistance.


    Footnotes
 
{ddagger} Present address: University of Wales College of Medicine, Medical Biochemistry & Immunology Department, Complement Biology Group, Heath Park, Cardiff CF14 4XN, UK Back


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 Materials and Methods
 Results
 Discussion
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