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Mol Cell Biol. May 2005; 25(9): 3814–3830.
PMCID: PMC1084281

The Human Stress-Activated Protein kin17 Belongs to the Multiprotein DNA Replication Complex and Associates In Vivo with Mammalian Replication Origins

Abstract

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G1/S border and throughout the S phase and was negligible in both G0 and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase α. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an ~600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

The kin17 protein was initially identified based on the cross-reacting property of antibodies raised against the stress-activated Escherichia coli RecA protein. kin17 displays a common epitope with the RecA protein and shares 47% homology over a 40-residue stretch in the RecA C-terminal region (2). In RecA protein, this region is involved in the regulation of DNA binding and in the SOS response (33). kin17 is a 45-kDa nuclear protein conserved during evolution, ubiquitously expressed in mammals (31). The main features of kin17 are its abilities to (i) bind directly to chromosomal DNA in human cells (7) and to RNA in mouse germ cells (56), (ii) bind preferentially to curved DNA found at the hot spots of illegitimate recombination (45, 46), (iii) complement the functions of a bacterial nucleoid protein called H-NS which binds to curved DNA and controls gene expression (66), and (iv) be upregulated after UV and ionizing radiations (6, 7, 9, 32, 42). Recently, a large-scale proteomic study of the human spliceosome-associated factors identified kin17 protein among 96 novel proteins related to splicing/mRNA processing, transcription, and cell cycle regulation (57). A link between the presence of UV-induced DNA damage and the mouse KIN17 pathway in ΔXPA mouse cells has also been reported (9). Furthermore, the integrity of the human global genome repair has been shown to be a crucial step for upregulation of the human KIN17 gene after UV irradiation. In particular, the presence of functional XPA and XPC proteins is a prerequisite for the upregulation of human KIN17 gene expression after UV-C (41). Interestingly, XPA, XPC, and RPA proteins have been involved in DNA damage recognition (4).

Chromosomal proteins often interact with DNA to control maintenance, propagation, and expression of the genome. Despite the identification of an increasing number of proteins that are involved in DNA replication, recombination, and repair, the mechanisms of these processes and the overlaps between them remain to be elucidated in mammalian cells. Evidence involving the human stress-activated kin17 protein in some aspects of DNA replication is accumulating. Indeed, kin17 forms intranuclear foci and accumulates in the nuclei of proliferating cells (32). Strikingly, kin17 concentrated in large nuclear foci associated with RPA after gamma irradiation (7). Cells presenting low levels of this protein also showed a prolongation of the S phase of the cell cycle associated with an accumulation of cells in early and mid-S phase, a decreased rate of DNA synthesis, and an increased sensitivity to gamma irradiation (7, 17). Besides, we have reported a physical interaction between human kin17 and simian virus 40 (SV40) large T antigen leading to both in vitro and in vivo DNA synthesis inhibition (30, 47). This compelling evidence pointed to a link between kin17 and DNA synthesis. However, it remained unclear whether kin17 is involved in replication, repair, or some other aspects such as the remodeling of chromatin architecture which could alter the efficiency of DNA replication. Indeed, kin17 is present in all eucaryotes, suggesting conservation of function (31).

The identification and isolation of proteins interacting with origins of replication are essential for understanding the molecular mechanisms initiating DNA replication and preventing genome overreplication. Several authors suggested that nascent DNA and several proteins involved in DNA synthesis may be linked to the nonchromatin ribonucleoprotein network known as the nuclear matrix, thereby forming replication foci (5, 13). In addition, nonchromatin nuclear elements have also been implicated in the initiation step of DNA replication, and putative origins of replication are often situated close to matrix attachment regions, which are believed to connect chromatin loops to the nuclear matrix (5, 13, 14, 16, 20, 21, 38), modulating chromatin architecture to facilitate the activity of origins of replication (53).

To further define the contribution of kin17 to DNA replication, we recently found an increased association of kin17 with the nuclear matrix in S-phase HeLa cells (48) and in mouse germ cells (56). Besides, cells entering into S phase exhibited an increased kin17 protein level which colocalized with active sites of DNA synthesis, as evidenced by immunoelectron microscopy of bromodeoxyuridine pulse-labeled cells (8). In the present work, we reinforce these data by showing that ultrastructural localization of endogenous kin17 protein in human cells also overlapped the specific stainings of RPA70, PCNA, and DNA polymerase α. For the first time, we demonstrate that kin17 protein associates in vivo specifically with both monkey and human DNA fragments containing replication origins at the G1/S boundary and throughout the S phase. We used the limited formaldehyde cross-linking technique (65), followed by real-time PCR of the immunoprecipitated kin17 protein-DNA cross-links as template. Furthermore, anti-kin17 antibodies hampered in vitro DNA replication of a plasmid containing a minimal replication origin and addition of recombinant kin17 protein partially restored the activity; this result emphasized the involvement of kin17 protein in mammalian DNA replication. To determine the possible interaction of kin17 protein with the DNA replication apparatus, we then performed biochemical fractionation of nuclear extracts (NE) by using two different methods in order to isolate multiprotein DNA replication-competent (RC) complexes. We showed that kin17 copurified with multiprotein replication complexes (MRCs) and is a necessary component of these complexes for efficient DNA replication activities. Taken together, we now characterize kin17 protein as (i) a stress-activated protein immunorelated to the bacterial RecA protein, (ii) a component of the MRC, and (iii) a protein tightly associated in vivo with mammalian DNA replication origins. In considering our latter results, it seems likely that kin17 protein could create a link between functional replication factories, origins of replication, and DNA damage recognition.

MATERIALS AND METHODS

Cell cultures and synchronization.

Human cells growing either in suspension (cervical carcinoma HeLa S3) or in monolayers (HeLa and colorectal carcinoma RKO) were maintained at 37°C in Dulbecco's modified Eagle's medium (Gibco). Monkey CV-1 cells growing in monolayers were maintained at 37°C in minimal essential medium α (Invitrogen). All media were supplemented with 10% fetal calf serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin under 5% CO2. To determine the association with mammalian replication origins, HeLa S3 cells were synchronized as follows. For synchronization to the G0/G1 phase, 80% confluent cells were placed in serum-free medium for 48 h; for synchronization to G1/S, S, and M phases, 40% confluent cells were treated with 2 mM thymidine for 12 h and then released for 9 h in regular medium without thymidine and subsequently incubated for 12 h with 2 mM thymidine and 400 μM mimosine. For S-phase synchronization, the cells were released from the thymidine/mimosine block for 2 h and 4 h in regular medium; for synchronization to M phase, the cells were released from the thymidine/mimosine block in regular medium supplemented with 1 μg/ml of nocodazole for 14 h. Cell synchronization was monitored by flow cytometry. For the isolation of the RC complex, HeLa S3 cells were synchronized at the G1/S border by growing cells with 2 mM thymidine as described by Stein and Stein (64). After thymidine removal and being twice washed with Dulbecco's modified Eagle's medium, cells were released from their arrested state by adding fresh culture medium for 2 h. The progression of these cells toward the cell cycle was analyzed by flow cytometry as described elsewhere (8) and was 10.1% in G1 phase, 60.2% in S phase, and 29.6% in G2/M phase. Cells were then harvested and washed three times with phosphate-buffered saline (PBS). Cells were pelleted by low-speed centrifugation, and the cell pellets stored at −80°C until use.

Sample analysis and antibodies.

Immunoblotting and preparation and characterization of mouse monoclonal antibodies K36 and K58 against human kin17 protein were described previously (7, 47). Purified monoclonal antibodies K36 and K58 were used at a concentration of 40 ng/ml. Other antibodies used were monoclonal anti-human PCNA (clone PC10; 50 ng/ml; Novo Castra), monoclonal anti-human RPA70 (clone NA13; 50 ng/ml; Oncogene Research Products), monoclonal anti-human cyclin A (clone CY-A1; 1/1,000; Sigma), monoclonal anti-human RFC p140 (1/1,000; gift from B. Stillman, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), polyclonal anti-calf thymus catalytic subunit DNA polymerase α (1/1,000; gift from A. M. Holmes). For immunoelectron microscopy labeling, we used a polyclonal anti-PCNA directed and purified against PCNA of Schizosaccharomyces pombe origin (1/10; gift from I. Tratner, Institut Curie-CNRS UMR 2027, Orsay, France) and the SJK132-20 monoclonal anti-DNA polymerase α (1/10; gift from S. Linn, Berkeley, CA). Peroxidase-labeled goat anti-mouse monoclonal antibody binding (1/10,000; Jackson Laboratories) was visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). Alkaline phosphatase-labeled anti-mouse monoclonal antibody binding (1/10,000; Sigma) was visualized using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (BCIP) tablets (Roche).

Immunoelectron microscopy detection of kin17 with replication proteins.

For fixation and embedding, specimens were processed as described elsewhere (8, 61). Cells were fixed for 1 h at 4°C with either 4% formaldehyde (Merck, Darmstadt, Germany) in 0.1 M Sörensen phosphate buffer (pH 7.3) or 1.6% glutaraldehyde (Taab Lab. Equip. Ltd., Reading, United Kingdom) in the same buffer. During fixation, the cells were scraped from the plastic substratum and centrifuged. Pellets were dehydrated in increasing concentrations of methanol and embedded in Lowicryl K4 M (Chemische Werke Lowi, Waldkraiburg, Germany) at low temperature according to Roth (59). Polymerization was carried out for 5 days at −30°C under long-wavelength UV light (TL 6W Philips fluorescent tubes). Lowicryl thin sections of cells were first placed for 2 min over drops of bovine serum albumin (BSA; 5% in PBS) in order to prevent background. For double labeling of the human kin17 protein with DNA polymerase α and RPA, ultrathin sections of Lowicryl-embedded material were collected on gold grids (600 mesh) without Formvar. For PCNA, gold grids (300 mesh) were used with Formvar. On one face, sections of cells were floated for 1 h at room temperature on drops of mouse monoclonal anti-kin17 protein antibodies (a mix of monoclonal antibodies K36 and K58) diluted 1/10 in PBS or over normal mouse serum diluted 1/10 in PBS as control, and kin17 protein was revealed with goat anti-mouse immunoglobulin G (IgG) conjugated to gold particles (Biocell Research Laboratories, Cardiff, United Kingdom) 5 nm in diameter diluted 1/30 in PBS. On the other face of the grid, immunolabeling was carried out with mouse monoclonal anti-RPA70, anti-DNA polymerase α, or over normal mouse serum as control, all diluted 1/10 in PBS. Double labeling of kin17 and PCNA proteins using polyclonal anti-PCNA was carried out on the same face of the grid. Proteins were revealed with goat anti-mouse or goat anti-rabbit IgG conjugated to gold particles 10 nm in diameter, diluted 1/30 in PBS. After washing, grids were rapidly rinsed in a jet of distilled water, air dried, and stained for 10 min with 5% aqueous uranyl acetate. Grids were observed with a Philips 400 transmission electron microscope at 80,000 eV. To control the labeling procedure on each face, RPA70 or DNA polymerase α were first immunolabeled as above on one face of the grid, and after being air dried the other face was floated with goat anti-mouse IgG conjugated to 5-nm gold particles. In these conditions, very few 5-nm gold particles could be visualized (data not shown).

Association with mammalian DNA replication origins. (i) In vivo cross-linking.

Cross-linking was performed as described by Ritzi et al. (58), with minor modifications. In brief, 80% confluent CV-1 and HeLa cells grown as described above were washed twice with PBS and then formaldehyde (1%) in warm medium without serum was added for 10 min. Cells were then lysed (at 4°C) in lysis buffer (50 mM HEPES/KOH [pH 7.5], 140 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, one capsule of protease inhibitors; Roche Molecular Biochemicals) and drawn into and out of a 21-gauge hypodermic needle three times to lyse cells and disperse the nuclei. Cell lysates were then layered over 4 ml of 12.5% glycerol in lysis buffer, and nuclei were pelleted by spinning at 750 × g for 5 min in a benchtop centrifuge. The nuclear pellet was resuspended in 1 ml of lysis buffer.

(ii) Chromatin fragmentation.

Cross-linked or non-cross-linked nuclei were sonicated 10 times for 30 s each time, and the chromatin size was monitored by electrophoresis (27). This treatment generated fragments of ~20 kb. To further reduce the chromatin size into smaller fragments of 1.5 to 3.5 kb, DNA was then digested with SphI, HindIII, PstI, and EcoRI restriction endonucleases in NEB2 buffer (100 U of each; New England Biolabs, Beverly, MA) at 37°C for 6 h.

(iii) Immunoprecipitation and DNA isolation.

Sheared chromatin extracts were incubated first with 50 μl of protein G-agarose (Roche Molecular Biochemicals). These cleared chromatin lysates were incubated at 4°C for 6 h on a rocker platform with either 50 μl of normal rabbit serum (NRS; Santa Cruz Biotechnology, Santa Cruz, CA) or 20 μg of antibodies directed against kin17 (purified IgG K36 or K58) or NF-κB p65 (goat polyclonal antibody C-20; Santa Cruz Biotechnology). Protein G-agarose (50 μl) was then added, and the incubation was continued for 12 h. The precipitates were successively washed twice for 5 min with 1 ml of each buffer: lysis buffer, WB1 (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% sodium deoxycholate), WB2 (as WB1 with no NaCl), and 1 ml of TE (20 mM Tris-HCl [pH 8.0], 1 mM EDTA). The precipitates were resuspended in 200 μl of extraction buffer (1% sodium dodecyl sulfate [SDS]/TE). Half of the sample was then incubated at 65°C overnight to reverse the protein/DNA cross-links, followed by 2-h incubation at 37°C with 100 μg of proteinase K (Roche Molecular Biochemicals). The other half (nonreversed cross-link) was incubated at 50°C for 1 h with 100 μg of proteinase K. Finally, DNA was purified using QIAquick PCR purification columns (QIAGEN, Valencia, CA).

(iv) Real-time PCR quantification analysis of immunoprecipitated DNA.

PCRs were carried out in 20 μl with 1/200 of the immunoprecipitated material using LightCycler capillaries (Roche Molecular Biochemicals) and LightCycler-FastStart DNA Master SYBR green I (Roche Molecular Biochemicals). PCR mixtures contained 3 mM MgCl2 and 1 μM each appropriate primer. Primer sets ors8 (monkey) and lamin B2 (LB2; human) amplify a 150-bp or a 232-bp fragment, respectively, in origin-containing sequences. As controls, primer sets CD4 intron (monkey) and C1 (human) amplify a 258-bp or a 240-bp fragment, respectively, in non-origin-containing sequences. Primers were designed as 20- to 22-mers with ~50% GC content as already published (34, 51). The quantification of PCR products was assessed by the LightCycler (Roche Molecular Biochemicals) using SYBR green I dye (55). Standardization was made with genomic DNA (9.3, 18.6, 27.9, 37.2, and 55.8 ng) obtained from total cell lysates of non-cross-linked logarithmic 80% confluent cells. The quantification program used a single fluorescence reading at the end of each elongation step. Arithmetic background subtraction was used, and the fluorescence channel was set to F1. Typically, an initial denaturation for 10 min at 95°C was followed by 35 cycles with denaturation for 15 s at 95°C, annealing for 10 s at 45°C (primer set ors8 150) or 50°C (primer set CD4 intron), and polymerization for 15 s at 72°C. The specificity of the amplified PCR products was assessed by performing a melting curve analysis cycle with a first segment set at 95°C for 0 s and a temperature transition of 20°C/s, a second segment set at 45°C or 50°C (depending on the annealing temperature of the primer set used) with a temperature transition rate of 20°C/s, and a third segment set at 95°C with a temperature transition rate set at 0.2°C/s. PCR products were also separated on 2% agarose gels, visualized with ethidium bromide (BET), and photographed with an Eagle Eye apparatus (Speed Light/BT Sciencetech-LT1000).

In vitro mammalian DNA replication assay.

In vitro DNA replication assays were performed as previously described, with some modifications (52). One hundred micrograms of total HeLa cell extract was incubated with various amounts of anti-kin17 antibody (K36 or K58), recombinant human kin17 protein, NRS, or hypotonic solution (20 mM HEPES [pH 7.8], 5 mM K acetate, 0.5 mM MgCl2, 0.5 mM dithiothreitol [DTT]) for 20 min on ice. This mixture was used to replicate in vitro 150 ng of p186 plasmid DNA (67). As an internal control for differences in DNA recovery and completeness of the DpnI digestion, we included unmethylated pBluescript KS+ in each reaction mixture. One-third of the replication products were digested with 1.5 U of DpnI for 60 min. Both undigested and digested products were resolved by electrophoresis in a 1% agarose gel in 1× TAE buffer at 50 V for 15 h; then the dried gel was exposed to an imaging plate for 6 h, and the DpnI-resistant bands (forms II and III) were quantified by densitometric measurements using Image Gauche (Fuji Photo Film Co., Ltd.). The results were normalized for the amount of DNA recovered from the in vitro replication assay and for the amount radionucleotide incorporated in the unmethylated pBluescript KS+ propagated in dam(−) bacterial cells. This incorporation was due to DNA repair, since this plasmid did not contain a mammalian origin of DNA replication. Also, the unmethylated plasmid cannot be digested by DpnI, since DpnI cleaves only fully methylated DNA. In addition, a reaction with methylated pBR322, a plasmid that does not contain a mammalian origin of DNA replication, was also performed to show that the observed DpnI-resistant bands (forms II and III) were origin dependent. The total amount of radionucleotide incorporated was expressed as a percentage of the control reaction with hypotonic buffer.

NE fractionation.

HeLa S3 NE (CILBiotech, Mons, Belgium) prepared according to the protocol of Dignam et al. (19) were first loaded onto a 100-μl Mono Q column (Smart System; Amersham Pharmacia Biotech) equilibrated with 20 mM Tris-HCl (pH 8.0), 100 mM KCl, and 0.5 mM DTT and step eluted in 0.2 M KCl, 0.3 M KCl, and 1 M KCl, all in the equilibration buffer. Five fractions (100 μl each) were collected, and an equal volume from each fraction was loaded onto a 10% SDS-polyacrylamide gel, followed by Western blotting. Fractions 3 of the NE before fractionation, the flowthrough (FT), and those eluted in 0.3 M KCl and 1 M KCl were then applied to a Superose 6 HR 10/30 column (Smart System; bed volume, 2.4 ml; 40 μl/min, 4°C) equilibrated with 20 mM HEPES (pH 7.5), 0.5 mM DTT, 0.1 M KCl, 0.3 M KCl, or 1 M KCl. In some experiments, BET (50 μg/ml) was added to the NE before size exclusion chromatography. Fractions (40 μl) were collected, and equivalent volumes were loaded onto a 10% SDS-polyacrylamide gel, followed by Western blotting. Protein standards (apoferritin, β-amylase, ADH, BSA, and cytochrome c) were analyzed under the same conditions. The molecular mass of protein complexes was determined from the calibration curve of molecular masses of protein standards.

Isolation and analysis of the RC complex.

The RC complex was isolated as described by Frouin et al. (22).

Buffers.

Buffer A contained 1 mM KH2PO4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA (pH 8.0), 10% (vol/vol) glycerol, and 0.5 mM PMSF. Buffer B consisted of buffer A containing 0.35% (vol/vol) Triton X-100. Buffer C consisted of PBS containing 5 mM MgCl2, 1 mM EDTA (pH 8.0), 10% (vol/vol) glycerol, and 0.5 mM PMSF. Buffer D consisted of 50 mM Tris-HCl (pH 7.5), 0.35 M NaCl, 1 mM EDTA (pH 8.0), 1 mM DTT, 10% (vol/vol) glycerol, 1 mM PMSF, and 1/1,000 protease inhibitor cocktail. Buffer E consisted of buffer D containing 0.01% (vol/vol) Nonidet P-40. Buffer F consisted of 25 mM bis-Tris (pH 6.6), 50 mM NaCl, 0.5 mM ATP, 1 mM DTT, 5% (vol/vol) glycerol, and 1 mM PMSF. Buffer TDB consisted of 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 10 mM MgCl2, 2% (vol/vol) glycerol, and 0.2 mg/ml BSA.

Isolation of HeLa cell nuclei.

Cells (108) were resuspended in 1 ml of buffer A and lysed for 1 h on ice in 9 ml of buffer B. The suspension was centrifuged at 900 × g for 10 min at 4°C in a Sorvall RC-5B centrifuge, and the pellet containing the nuclei washed with 1.5 ml of buffer C and centrifuged at 900 × g for 10 min at 40°C.

Preparation of NE.

Nuclei were resuspended in 1 ml of buffer D containing 0.2 M NaCl, stirred for 30 min at 4°C, and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was kept as the 0.2 M NaCl NE. The pellet was resuspended in 1 ml of buffer E containing 0.35 M NaCl, stirred for 1 h at 4°C, and centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was kept as the 0.35 M NaCl NE.

Size exclusion chromatography.

Half a milliliter of 0.2 M NaCl NE from HeLa cells was loaded onto a Superdex 200 HR 10/30 gel filtration (GF) column (Pharmacia) equilibrated with buffer F. The column was previously calibrated with blue dextran (2,000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), ovalbumin (43 kDa), and myoglobulin (17.5 kDa) as molecular weight standards.

Mono S column chromatography.

GF fractions that contained the proteins of interest were loaded onto a Mono S column (Pharmacia) equilibrated with buffer F. Proteins were eluted with a nonlinear gradient of 0.1 to 1 M NaCl.

Heparin-Sepharose chromatography.

Active fractions from the Mono S column were pooled and loaded onto a HiTrap heparin-Sepharose column (Pharmacia) equilibrated with buffer F. The column was eluted with a linear gradient of 0.1 to 1 M NaCl.

Mono Q chromatography.

Active fractions from the heparin-Sepharose column were pooled and loaded onto a Mono Q column (Pharmacia) equilibrated with buffer F. The column was eluted with a linear gradient of 0.1 to 1 M NaCl.

Immunoblot analysis of column fractions (spot test).

Four microliters of each fraction was spotted onto a nitrocellulose membrane without electrophoretic separation, and the membrane was treated as in standard immunoblot protocols.

DNA polymerase α/δ activity assays.

A final volume of 25 μl contained the following components: buffer TDB, 8 μM [3H]dNTP (1.5 Ci/mmol), 10 mM MgCl2, and 0.5 μg of poly(dA)/poly(dT).

Native polyacrylamide gel electrophoresis.

GF fractions (20 μg) were resolved using precast 4 to 15% polyacrylamide gel (Bio-Rad) with the exclusion of SDS in the polyacrylamide gel, running and sample buffer as previously described (68). 2-Mercaptoethanol was excluded from the sample buffer. All samples were electrophoresed at 4°C for 16 h, 40 V. Proteins were electrophoretically transferred to nitrocellulose at 12 V for 16 h at 4°C. Immunoblotting was carried out as described previously. Thyroglobulin (669 kDa) and apoferritin (440 kDa), used as molecular weight standards (Pharmacia), were run on a parallel gel, and proteins were revealed by zinc staining (Bio-Rad).

Isolation and analysis of the MRC. (i) Preparation of NE.

HeLa cells were fractionated as described by Wu et al. (73). Briefly, 2 g of frozen HeLa cells was thawed and resuspended in 3 volumes of buffer containing 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 5 mM MgCl2, 0.1 mM PMSF, and 1 mM DTT. The resuspended cells were homogenized with a loose-fitting Dounce homogenizer. The homogenate was then subjected to a series of centrifugation steps to obtain postmicrosomal and nuclear fractions. The crude NE was resuspended in 2 volumes of a buffer containing 50 mM Tris-HCl (pH 7.5), 0.15 M KCl, 5 mM each EDTA and EGTA, 0.1 mM PMSF, and 1 mM DTT and gently stirred for 2 h at 4°C. The extracted nuclei were centrifuged at 100,000 × g for 1 h, and the supernatant was collected. The latter supernatant and the postmicrosomal supernatant were pooled and brought to 2 M in KCl. Solid polyethylene glycol (PEG 8000) was added to a final concentration of 5%, and the mixture stirred gently for 1 h at 4°C. PEG-precipitated material was pelleted by centrifugation for 30 min at 16,000 × g, and the supernatant was dialyzed for 3 h against two changes of a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, and 50 mM KCl. The dialysate was clarified by centrifugation for 10 min at 13,000 × g. The clarified fraction was layered over a 2 M sucrose cushion and subjected to centrifugation at 100,000 × g for 18 h at 4°C. The material above the sucrose interphase was collected and designated the HS-4 fraction (8/10 of the total). The sucrose interphase fraction was collected and designated HSP-4 (2/10 of the total). The HSP-4 fraction was dialyzed for 3 h against two changes of a buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 10% glycerol, and 1 mM DTT.

(ii) Size exclusion chromatography analysis.

The dialysate (in 50 mM Tris-HCl [pH 7.5], 0.5 M KCl, 1 mM EDTA, and 1 mM DTT) was subjected to size exclusion chromatography on a Superose 6 HR 10/30 column equilibrated in the same buffer. After GF, proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis for Western blot analysis. In some experiments, DNase I (400 U/ml) and RNase A (100 μg/ml) were added for 1 h at 4°C to the HSP-4 fraction before size exclusion chromatography. The column was calibrated as described earlier.

(iii) Mono Q chromatography.

To further purify the HSP-4 fraction, it was dialyzed and loaded onto a Mono Q column pre-equilibrated in dialysis buffer. The column was washed with 10 column volumes of the dialysis buffer, and the bound proteins were then eluted by 10 column volumes of the buffer adjusted to 1 M KCl. An aliquot of each of the collected fractions was saved for immunoblotting, DNA polymerase α, and ATPase activity analyses. The pooled collected fractions designated the high-salt eluate were dialyzed into 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, and 10% glycerol and stored in aliquots at −80°C.

DNA polymerase α activity assay.

DNA polymerase activity was tested as described previously (15) in 60 μl of buffer containing 10 mM Tris-HCl (pH 7.5); 5 mM MgCl2; 7.5 mM DTT; 50 μg/ml BSA; 0.5 μg of DNase I-activated calf thymus DNA; 25 μM each dATP, dCTP, and dGTP; 5 μCi of [α-32P]dCTP (3,000 Ci/mmol). Incorporation of radiolabeled nucleotides was determined by trichloroacetic acid precipitation.

ATPase activity assay.

ATPase activity of the HSP-4 and Mono-Q high-salt eluate fractions were measured as described previously (28) in 50 μl of ATPase buffer (30 mM NaCl, 5 mM KCl, 10 mM MgCl2, 10% glycerol, 2 mM β-mercaptoethanol in 20 mM Tris-HCl [pH 7.4]) containing 1 μg of heat-denatured calf thymus DNA, 200 pmol of unlabeled ATP, and 1 pmol of [γ-32P]ATP (3,000 Ci/mmol). The samples were incubated at 37°C for 10 min, and 700 μl of 7% activated charcoal (Sigma) in 50 mM HCl-5 mM H3PO4 was then added. The charcoal was precipitated by centrifugation (10 min at 11,000 × g) to remove unreacted ATP. The supernatant (100 μl) was mixed with scintillation cocktail and counted.

SV40 in vitro DNA replication assay.

The SV40 in vitro DNA replication assay was performed as described in the manufacturer's instructions (kit from Molecular Biology Resources, Milwaukee, WI). Briefly, extracts from HeLa cells (350 μg) in replication buffer (30 mM HEPES [pH 7.5]; 0.5 mM DTT; 4 mM ATP; 50 μM each CTP, GTP, and UTP; 100 μM each dATP, dGTP, dTTP, and dCTP; 7 mM MgCl2; 0.625 U of creatine phosphokinase; 40 mM phosphocreatine) were supplemented with 50 ng of SV40 origin-containing plasmid pUC.HSO or control plasmid devoid of SV40 origin pUC.8-4, 1 μg of purified T antigen, and 1 μCi of [α-32P]dCTP (3,000 Ci/mmol) (36). Where indicated, HeLa cell extracts were replaced with 20 μg of the HSP-4 fraction obtained as described below. The mixtures (final volume, 25 μl) were incubated at 37°C for 4 h. The reaction was stopped by addition of 1% SDS, 10 mM EDTA, and 100 μg/ml proteinase K. Following a 30-min incubation at 37°C and phenol-chloroform extraction, DNA was electrophoresed on a 1% agarose gel in 1× TAE at 55 V. Gels were dried, and DNA replication products were visualized by exposure to a PhosphorImager screen. To quantify DNA replication activity, one-fifth of the reaction mixture was removed at the end of the incubation period and mixed with 50 μl of yeast RNA coprecipitant and 1 ml of 10% trichloroacetic acid for 10 min. The content of the reaction tube was filtered through a Whatman GF/C filter disk on a vacuum filter apparatus. The disk was thoroughly washed with 10% trichloroacetic acid, followed by 95% ethanol, and transferred to a vial with scintillation fluid to count radioactivity.

RESULTS

Ultrastructural colocalization of kin17 with DNA replication proteins.

We have previously shown that the human kin17 protein accumulates in the nuclei of proliferating cells with predominant colocalization with active DNA replication sites by immunoelectron microscopy (8). Using a similar approach, we determined the colocalization of kin17 and other proteins belonging to the MRC. We used asynchronous proliferating human RKO cells, and we focused on RPA, PCNA, and DNA polymerase α. Dual labeling was carried out against either kin17/RPA70 (Fig. (Fig.1A),1A), kin17/PCNA (Fig. (Fig.1B),1B), or kin17/DNA polymerase α (Fig. (Fig.1C).1C). For kin17 staining, we used a mixture of two monoclonal antibodies (purified IgG K36 and K58) known to recognize distinct epitopes of the recombinant kin17 protein. kin17 protein was revealed with 5-nm-diameter gold particles, and the other proteins were revealed with 10-nm particles. We show three representative images obtained with RKO cells. kin17 protein was detected in foci scattered throughout the nucleoplasm, and most of them colocalized with RPA70, PCNA, and DNA polymerase α (Fig. 1A, B, and C). In particular, the overlapping staining of kin17 and RPA70 was striking. Because the ultrastructural colocalization of RPA, PCNA, and DNA polymerase α at sites of DNA synthesis has been previously demonstrated, it is likely that these foci containing kin17 correspond to DNA replication domains.

FIG. 1.
Ultrastructural colocalization of kin17 protein with DNA replication proteins. Ultrathin sections were prepared from proliferating RKO cells for immunoelectron microscopy as indicated in Materials and Methods. kin17 protein was identified with a mixture ...

kin17 takes part in nuclear protein complexes of high molecular weight.

We previously determined that ~60% (~20,000 molecules per cell) of the total cellular content of kin17 protein is present in nuclear foci upon treatment of HeLa cells with 0.1% Triton X-100 (48). The nuclear localization of kin17 increases in S phase of the cell cycle, and a fraction of it was resistant to high-salt and nuclease extractions. Previous data showed that kin17 interacts with distinct components of the nuclear structures, such as the nuclear matrix and the chromatin DNA, ex vivo and in vivo (48, 56). We tested whether the human kin17 protein could reside in distinct protein complexes in the nucleus. Therefore, we performed a fractionation of HeLa NE using a Superose 6 gel filtration column before and after a Mono Q anion-exchange column. Size exclusion chromatography of NE demonstrated that kin17 was present in different complexes of high molecular weight [Fig. [Fig.2A,2A, BET(−)]. Indeed, kin17 was eluted in the void volume (>2 MDa, fractions 2 to 4) and in fractions corresponding to ~600 kDa (fractions 15 to 18). The elution profile of PCNA and RPA was very similar to that of kin17, except for RPA, which was not detected in the void volume. We confirmed a previously reported observation that, in vivo kin17 was never observed in eluted fractions corresponding to monomers (8). To rule out the possibility that kin17-containing complexes were organized by DNA or RNA instead of protein-protein interactions, we treated the NE with both DNase I and RNase A or ethidium bromide before performing GF, as already described (35). Under these conditions, we failed to detect a significant difference in the elution profile of untreated [Fig. [Fig.2A,2A, BET(−)] and treated [Fig. [Fig.2A,2A, BET(+)] NE.

FIG. 2.
kin17 protein is a component of high-molecular-weight nuclear protein complexes. (A) Immunoblot analysis and chromatographic profiles of HeLa S3 NE fractionated on a Superose 6 GF column in the absence [BET(−)] and in the presence [BET(+)] ...

To determine whether kin17 and its coeluted proteins are associated within the same high-molecular-weight protein complexes, we performed a two-step fractionation of the NE using anion-exchange chromatography (Mono Q column), followed by size exclusion chromatography (Superose 6 column) of eluted fractions containing kin17. A fraction of kin17 bound to the Mono Q column eluted at 0.3 M and 1 M KCl (Fig. (Fig.2B).2B). According to the basic properties of kin17 (pI = 9.2), a substantial amount of this protein did not bind to the column and eluted in the FT and 0.2 M KCl fractions.

The fractions isolated after anion-exchange chromatography (FT 3, 0.3 M KCl fraction 3, and 1 M KCl fraction 3) were further characterized by size exclusion chromatography (Fig. (Fig.2C).2C). Gel filtration analysis and anion-exchange chromatography were run with the same elution buffer. In the FT fraction, kin17 protein eluted completely as a monomer (45 kDa) while only a part of it behaved as a monomer in the 0.3 M KCl fraction. Interestingly, RPA coeluted with kin17 in the 0.3 M KCl fraction (Fig. (Fig.2C).2C). In the 1 M KCl fraction, kin17 protein, PCNA, and RPA coeluted in high-molecular-weight protein complexes. This result indicates that the kin17-containing complex was ~600 kDa. It was noteworthy that the presence of kin17, RPA, and PCNA in the ~600-kDa complex was not modified with increasing ionic strength. In opposition, kin17 belonging to the >2-MDa complex was not detected after Mono Q column chromatography (Fig. (Fig.2A2A versus C). Altogether, these results indicate that a fraction of kin17 coeluted with RPA and PCNA proteins at a molecular mass of ~600 kDa, corresponding to multiprotein complex, in agreement with immunoelectron microscopy analysis.

kin17 protein copurifies with DNA replication complexes.

We asked whether kin17 protein may be detected among the proteins that copurify with the replication protein complex. Therefore, we used two different classical protocols leading to the isolation of discrete multiprotein complexes able to replicate DNA (73, 22). In a first step, proteins from HeLa cells were fractionated according to the protocol developed in the laboratory of Linda Malkas (73). This results in substantial purification of the DNA replication complex (MRC) (73). Briefly, extracted proteins were centrifuged onto a 2 M sucrose cushion and divided into an interphase fraction consisting of high-molecular-weight protein complexes (including the MRC referred to as fraction HSP-4) and a supernatant fraction consisting of soluble proteins (fraction HS-4). Figure Figure3A3A shows that the kin17 protein was found in the HSP-4 complex (fractions 9 to 10), suggesting that the kin17 protein might be a part of the MRC. The HSP-4 fraction was further purified by Mono-Q chromatography. RPA and kin17 proteins (fraction 6) were eluted with a similar elution profile, while PCNA was eluted at a higher salt concentration (Fig. (Fig.3B).3B). Moreover, as for kin17 protein and RPA, the highest DNA polymerase α and ATPase activities contained in the HSP-4 fraction eluted in fractions 6 and 7 (Fig. (Fig.3B3B).

FIG. 3.
kin17 protein is a component of the MRC in human HeLa cells. (A) Immunoblot analysis for the presence of kin17 protein in fractions from purification of the human MRC. Extracted nuclear proteins were centrifuged onto a 2 M sucrose cushion and divided ...

Eluted fractions 6 to 10 of the Mono Q column were then pooled, and after dialysis the molecular weight of the isolated protein complex was estimated by size exclusion chromatography. We observed that the protein complex of ~600 kDa containing RPA, PCNA, and kin17 protein was maintained during GF analysis (Fig. (Fig.3C).3C). Moreover, DNA polymerase α and ATPase activities were also found to coelute with kin17 protein, RPA, and PCNA (Fig. (Fig.3C).3C). To exclude the possibility that the kin17 protein contained in this fraction could be bound to nucleic acids rather than to a protein complex, we treated the HSP-4 fraction with both DNase I and RNase A before GF analysis. The elution profile of kin17 protein, as detected by Western blotting, and DNA polymerase α and ATPase activities were not modified by nuclease treatment, suggesting that kin17 participates within the complex through protein-protein interactions (Fig. (Fig.3D3D).

To further analyze the copurification of kin17 protein with a RC complex, we used a second protocol developed by Frouin et al. (22). This protocol includes size exclusion chromatography of NE from synchronized S-phase HeLa cells at a ionic strength of 0.2 M NaCl, followed by sequential Mono S, heparin, and Mono Q chromatography steps. The presence of the different proteins involved in DNA replication and cell cycle control was followed throughout the purification by immunoblot analysis and DNA polymerase activity. As shown in Fig. Fig.4A,4A, the kin17 protein coeluted with DNA replication proteins (RFC, polymerase α) and cell cycle regulation factors such as cyclin A with a retention volume of 6 ml, corresponding to a protein complex with an apparent molecular mass of >669 kDa. When polymerase α was assayed, the peak of activity was detected in fraction 6, matching the elution profile of DNA polymerase α (Fig. (Fig.4A).4A). Although the same protein profile was obtained with asynchronous HeLa cells (data not shown), an increased level of kin17 protein was detected by Western blotting in the fractions containing the peak of DNA polymerase α isolated from S-phase cells (Fig. (Fig.4B4B).

FIG.4.FIG.4.
kin17 protein copurifies with an RC complex from S-phase HeLa cells. (A) GF elution profile. Dot blot analysis of GF fractions of the 0.2 M NaCl NE from HeLa cells was performed with antibodies against polymerase α (pol α), RFC, cyclin ...

The peak of polymerase α activity (fractions 5 to 9) was pooled and loaded onto a Mono S column, and proteins were eluted with a linear gradient of 0.1 M to 1 M NaCl. Immunoblot analysis revealed that the peaks of polymerase α, cyclin A, RFC, and kin17 proteins eluted between 0.5 and 0.6 M NaCl (Fig. (Fig.4C).4C). Polymerase activity peaked in fraction 20. Fractions 16 to 30 were pooled and loaded onto a heparin-Sepharose column. Proteins were eluted using a linear gradient of NaCl of 0.1 to 1 M. DNA polymerase α, RFC, and cyclin A, revealed by immunoblot analysis, coeluted in fractions 30 to 39 (Fig. (Fig.3D),3D), and polymerase activity peaked in fraction 36 (Fig. (Fig.4D4D).

Finally, the active fractions from heparin-Sepharose were pooled (fractions 29 to 37) and loaded onto a Mono Q column. As shown in Fig. Fig.4E,4E, polymerase α, RFC, cyclin A, and kin17 coeluted in fractions 15 to 16. Polymerase activity peaked in fraction 16. Using this purification procedure, polymerase specific activity was enriched by more than 350-fold (data not shown). Peak fractions from each chromatographic step were analyzed by Western blotting using anti-kin17 and anti-RFC antibodies (Fig. (Fig.4F).4F). As shown previously, this multiprotein complex containing polymerase α, RFC, cyclin A, and kin17 protein isolated from S-phase HeLa cell NE is a relatively stable entity which survived successive purification steps.

In order to confirm the presence of the kin17 protein in a complex with replication and cell cycle regulatory factors, aliquots of fraction 6 of GF and fraction 16 of the Mono Q column were electrophoresed through a 4 to 15% native polyacrylamide gel, followed by immunoblotting with antibodies directed against the kin17 protein (Fig. (Fig.4G).4G). Anti-kin17 antibodies stained a band of >669 kDa corresponding to the RC complex (22). These results show that kin17 copurifies through four different chromatographic steps with DNA replication proteins and cell cycle regulatory factors as high-molecular-weight multiprotein complexes.

Cross-linking of kin17 protein with the RC complex to chromatin in vivo.

To investigate whether kin17 was associated with the chromatin-bound form of the RC complex, we performed in vivo cross-linking of proteins to DNA with formaldehyde in S-phase-synchronized HeLa cells as described previously (7, 22). Our approach was based on a cross-linking procedure that minimizes the formation of nonspecific cross-linking and largely excludes contamination with non-cross-linked material (25). Using this method, we previously showed that kin17 binds directly to chromatin DNA within protein-DNA complexes in HeLa cells (7). These complexes have a density of 1.4 g/ml, corresponding to a mass ratio of 1:1, characteristic of native chromatin (26). The complex isolated from 0.2 M NaCl NE (Fig. 4F and G) (22) probably represents a fraction that is not stably associated with the chromatin. For example, PCNA was not found associated with the soluble form of the RC complex but was found within the complex bound to the chromatin (22). To determine whether the fraction of kin17 that was present in the soluble form of the RC complex could be cross-linked to DNA, immunoblot analysis of free proteins and DNA/protein complex fractions was performed. We showed that 40% of kin17 and 70% of RPA proteins were present in DNA-protein complexes (Fig. (Fig.4H4H).

Purified DNA replication complex (HSP-4 fraction) immunodepleted of kin17 protein exhibited reduced replication activity in vitro.

In view of the copurification of kin17 with the MRC and the observed association of this protein with sites of active DNA synthesis, we tested the effect of the immunodepletion (Id) of the MRC (HSP-4 chromatographic fraction) with protein A-Sepharose beads coated with anti-kin17 K36 and K58 (1 μg of each), anti-PCNA clone PC10 (2 μg), and 2 μg of a mixture of IgG1 and IgG2b monoclonal antibodies used as an isotype control on the in vitro SV40 origin- and large T antigen-dependent DNA replication. After 1 h on ice, beads were pelleted and the supernatants were used for the in vitro replication assay. A representative autoradiogram of the in vitro replication experiment with the HSP-4 fraction (Fig. (Fig.5A,5A, lane 1) and with the kin17-immunodepleted HSP-4 fraction (Fig. (Fig.5A,5A, lane 3) is shown. The Id of kin17 protein decreased the level of in vitro DNA replication of 60% compared to control reaction, in which the HSP-4 fraction was preincubated with the same amount of protein A-beads and buffer. In a second set of experiment, an inhibition of DNA replication of 45% was obtained with the kin17-immunodepleted HSP-4 fraction compared with mock-immunodepleted extract with isotype control antibodies and reached 75% inhibition with PCNA-immunodepleted extract, used as a positive control (Fig. (Fig.5B).5B). Virtually all PCNA and kin17 protein was depleted from the HSP-4 fraction, as shown by the absence of a signal in the immunodepleted fraction (Fig. (Fig.5C),5C), while the levels of the other proteins remained unaffected. These results suggest that the presence of kin17, acting either alone or in concert with replication proteins interacting with kin17, is necessary for an optimal process of DNA replication.

FIG.5.
Immunodepletion of kin17 protein inhibits in vitro replication of SV40 origin- and large T antigen-dependent DNA replication. The DNA replication activity of immunodepleted and mock-immunodepleted HSP-4 fraction for the kin17 protein was assessed by the ...

kin17 is tightly associated in vivo with mammalian DNA replication origins.

Since kin17 was clearly associated with the DNA replication apparatus and tightly associated with nuclear structures (chromatin DNA and nuclear matrix) (48, 56), we investigated a potential role of this protein in the regulation of DNA replication by binding at origins of replication. To analyze whether kin17 preferentially binds in vivo to DNA sequences containing origins of replication, chromatin immunoprecipitation assays were performed as previously described (50). Either CV-1 or HeLa cells were treated in vivo with formaldehyde to cross-link DNA-binding proteins, and kin17 was immunoprecipitated with anti-kin17 antibodies. Immunoprecipitation was performed with two different monoclonal anti-kin17 antibodies (K36 or K58) or control serum (NRS). The DNA immunoprecipitated by anti-kin17 antibodies was enriched in origin-containing sequences. This association was quantified by real-time PCR (Roche Molecular Biochemicals). Specific primer sets for either origin-containing sequences ors8 (monkey) and LB2 (human) or non-origin-containing sequences CD4 (monkey) and C1 (human) were used. The association of kin17 protein with the origin-containing ors8 sequence in exponentially growing CV-1-cross-linked cells was approximately 12- to 18-fold higher than in NRS-immunoprecipitated DNA (Fig. (Fig.6A).6A). In comparison, the association of kin17 protein with the CD4 intron genomic region that does not contain an origin of DNA replication was ~15- to 20-fold lower than with the ors8 origin-containing region. In agreement with the results obtained with CV-1 cells, the association of kin17 protein with origin-containing region LB2 in exponentially growing HeLa cross-linked cells was ~10- to 17-fold higher than the amount immunoprecipitated with NRS (Fig. (Fig.6B).6B). In addition, the association of kin17 protein with the C1 genomic region, which does not contain an origin of DNA replication, was ~8- to 10-fold lower than with LB2 origin-containing region. Note that using DNA template corresponding to non-origin-containing regions, the amount of DNA immunoprecipitated with two different monoclonal anti-kin17 antibodies was similar to that with control serum (NRS; Fig. 6A and B), further confirming the specificity of the enrichment procedure.

FIG.6.
Direct involvement of kin17 protein in DNA replication. (A, B) kin17 protein associates in vivo with mammalian DNA replication origins. Quantification of DNA abundance in origin-containing and non-origin-containing sequences by real-time PCR is shown. ...

To analyze the cell cycle-dependent association of kin17 protein with DNA sequences containing origins of replication, HeLa S3 cells were synchronized in different phases of the cell cycle (G0, G1/S, S 2 h and 4 h after release, G2, and M phases) and chromatin immunoprecipitation assays were performed as for asynchronous cells with K36 and K58 antibodies. The results showed that the association of kin17 protein with origin-containing region LB2 is dependent of the cell cycle (Fig. (Fig.6C).6C). The association of kin17 protein with the LB2 region was 4- to 9-fold higher at the G1/S boundary, 8- to 10-fold higher in S phase 2 h after release, and 7- to 24-fold higher in S phase 4 h after release, by comparison with their association in G0 (serum-starved) cells (Fig. (Fig.6C).6C). Furthermore, there was no significant association of kin17 protein with the LB2 region during mitosis, as observed for cells in G0.

The amount of kin17 protein determined the replication efficiency of p186, a plasmid carrying a minimal mammalian origin of replication.

Since kin17 participates in the DNA replication apparatus and is associated in vivo with mammalian replication origins (ors8 and lamin B2; Fig. 6A and B) as a function of the cell cycle (Fig. (Fig.6C),6C), we evaluated the effects of kin17 protein on DNA replication activity in an in vitro replication system based on a plasmid carrying an endogenous replication origin (p186). The in vitro system used allows the dissection and study of the proteins required for DNA replication (43, 44, 50, 51).

We performed in vitro DNA replication assays using either cytoplasmic extract or NE from HeLa cells together with increasing (2 μg, 5 μg, and 10 μg) amounts of anti-kin17 antibodies (K36 or K58), as well as control serum (NRS; Fig. Fig.6D).6D). Addition of 10 μg of either the K36 or K58 anti-kin17 antibodies inhibited the relative in vitro replication of p186 by approximately threefold. In contrast, addition of NRS had no effect, indicating that the observed inhibition was specific to the binding of specific antibodies to kin17. When pBR322 was used as a DNA template control, which does not contain a mammalian origin of DNA replication, no DpnI-resistant products were obtained (data not shown), indicating that this plasmid did not replicate. Addition of 1 μg of recombinant human kin17 protein to 5 μg of each of the K36 and K58 anti-kin17 antibodies partially restored the relative in vitro replication of p186, indicating the requirement of kin17 protein to in vitro DNA replication activity.

DISCUSSION

The kin17 proteins are conserved among eucaryotes and are expressed in a broad range of tissues and cell types. In mammals, kin17 proteins are largely found in the nucleus and have been potentially involved in essential cellular processes, including cellular response to DNA damage, gene expression, and DNA replication. Previously, a role for kin17 in mammalian DNA replication has been suggested, since its depletion, by means of overexpression of a KIN17 antisense transcript in human cells entailed major growth disadvantages with an accumulation of cells in early and mid-S phase (7). We have recently observed that kin17 has both an increased expression in cells entering into S phase (8) and an increased association with the nuclear matrix in S phase (48). This interaction has been confirmed in vivo in mouse germ cells (56). The nuclear matrix is believed to be implicated in the initiation step of DNA replication. To further investigate the role of kin17 with DNA replication, we have localized kin17 protein within sites of DNA replication by means of immunoelectron microscopy of bromodeoxyuridine pulse-labeled cells (8). Strikingly, kin17 foci colocalized with active sites of DNA synthesis.

Now, we show the direct involvement of kin17 in DNA replication by demonstrating that kin17 protein (i) is associated with and involved in multiprotein DNA replication complexes and (ii) is able to recognize specific genomic regions containing origins of replication as a function of the cell cycle.

In mammalian cells, DNA replication is an essential and highly regulated process that must be achieved in a limited period of time before mitosis. This efficient process requires the coordinated activity of a variety of enzymes and accessory proteins. The basic mechanisms of DNA replication have been elucidated mostly by biochemical and genetic studies. The in vitro reconstitution of the complete process of plasmid DNA replication containing SV40 ori with individual purified proteins allows the identification of the minimal number of essential components (72). Accumulating in vivo evidence indicates the existence of large multiprotein complexes containing replication proteins that function to replicate DNA and may help to coordinate the replication activity with other essential process like mitosis (23, 39). Multiprotein complexes are groups of proteins that interact with each other at the same time and place, forming a single multimolecular machine. Examples of identified protein complexes include several large transcription factor complexes, the anaphase-promoting complex, RNA splicing and polyadenylation machinery, and protein export and transport complexes (62).

Several large macromolecular complexes of replication proteins called the DNA synthesome (11, 12, 39, 73) or replication-competent protein complex (RC complex) (22, 37) have been isolated from mammalian cells. The DNA synthesome protein complex (39) includes at least two different DNA polymerases (α and δ), DNA primase, DNA helicase, a single-stranded DNA binding protein trimer (RPA), a clamp loading factor (RFC), a polymerase clamp (PCNA), nucleases, DNA ligase I, PARP-1, and DNA topoisomerases I and II, which cooperate to replicate the DNA template (39). Recently, the development of an original protocol using mild nuclear extraction conditions, as well as buffer optimization (inclusion of ATP), allowed the preservation and purification of a high-molecular-weight RC complex (22, 37). The composition of this protein complex varies during the cell cycle, and it contains DNA replication proteins (DNA polymerases α and δ, RPA, RFC, DNA ligase I, PARP-1, and DNA topoisomerase I) associated with cell cycle regulation factors like cyclin A, cyclin B1, Cdk2, and Cdk1.

These two different biochemical protocols show for the first time that kin17 protein coelutes with DNA replication complexes after several chromatographic steps. The fractionation of the NE to isolate the RC complex involved GF, cationic (Mono S and heparin), and anionic (Mono Q) columns. The kin17 protein and the proteins involved in the RC complex were coeluted from the different columns using salt concentrations as high as 500 mM. So, spurious cofractionation of kin17 protein and proteins involved in DNA replication and cell cycle regulation throughout the purification protocol is unlikely. Although the exact protein compositions of the different DNA replication complexes isolated present variations (23, 39), most of their components are shared, thus arguing against extraction procedure differences.

At the cellular level, eukaryotic DNA replication takes place at microscopically visible subnuclear sites called replication foci (49) consisting of clusters of replisomes or synthesomes, each being large macromolecular complexes containing all the activities necessary for the complete duplication of one replicon. It is not known whether entire replisomes within one cluster move from one replicon to the next or whether they disassemble into individual proteins or into small complexes. It is also not known whether they were derived from the nucleoplasmic pool or from replication foci. Moreover, recent studies suggest that the DNA synthesome exists in two states, a chromatin-free state (which may be predominant at the G1/S transition) and a chromatin-bound state (which may be enriched during S phase) (22). For instance, although PCNA is present in the DNA synthesome, PCNA is not tightly bound to it (39). In addition, and unlike the human kin17 protein, PCNA was not found associated with the soluble form of the RC complex (22, 37). The RC complex probably represents a soluble or loosely DNA-associated form, and PCNA may remain bound to chromatin to tether the RC complex to DNA. Accordingly, Sporbert et al. revealed recently that the major fraction of PCNA was not preassembled in the nucleoplasm into large replication complexes by in situ extractions and fluorescence recovery after photobleaching experiments (63). But, PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery bound to chromatin throughout the replication of an entire replicon (63). Interestingly, kin17 protein in the RC complex was also found in cross-linked DNA-protein complexes and kin17 protein partitioned equally between free proteins and DNA-protein complexes. Taking into account that several enzymes expected to be involved in DNA repair, recombination, or chromatin remodeling were present on newly synthesized DNA (proteins not required for DNA synthesis per se), one can speculate that each replication factory could contain two pools of molecules, like PCNA and kin17, one engaged in DNA replication and the other constituting the reserve of recruited replication factors in the vicinity of active replicons, thus providing a link between synthesis and repair pathways.

We further demonstrate that kin17 protein is tightly associated with specific genomic regions containing replication origins (ors 8 and LB2) in comparison with non-origin-containing sequences (CD4 intron and C1). We used the well-characterized formaldehyde cross-linking approach (25, 29, 50, 51, 58, 65). Formaldehyde treatment of cells readily produces protein-protein and protein-DNA cross-linked complexes, and antibodies were then employed to immunoprecipitate kin17. The DNA recovered from the immunoprecipitates was analyzed by real-time PCR to quantitatively assess whether the recovered template DNA was enriched in origin-containing sequences. The in vivo association of kin17 with origins of replication was investigated with specific primer sets from the monkey origins ors8 (ors8 150) and the human origins LB2 (lamin B2). We show for the first time the specific association of kin17 with these origins, since DNA fragments recovered from the kin17 immunoprecipitates were enriched ~10-fold in origin-containing sequences compared with other portions of the genome (Fig. 6A and B). Furthermore, our results demonstrate that the association of kin17 protein with origin-containing sequences is specific to the G1/S and S phases of the cell cycle, suggesting its participation before origin firing to initiation and elongation phases of the DNA replication process. It should be noted that, in asynchronous cells, similar and even lower levels of enrichment of origin-containing sequences were obtained by chromatin immunoprecipitation using anti-Ku70 or anti-Ku86 (51, 60), anti-CBP (the human cruciform binding protein) (50), and anti-14-3-3 sigma (1) antibodies. A number of controls were included to ensure that the amplification signals obtained were due to specific protein/DNA interactions. As negative antibody controls, we used an antibody directed against the p65 subunit of transcription factor NF-κB (a DNA-binding protein not known to bind to origin DNA; data not shown) and NRS antibodies. The background signal arising from DNA that was immunoprecipitated nonspecifically by these antibodies was quantified and estimated to be less than 15% in origin-containing sequences (ors8 and lamin B2), as well as non-origin-containing sequences (Fig. 6A and B).

The same anti-kin17 antibodies used in the chromatin immunoprecipitation assays were also used in an in vitro replication assay to investigate their effect on in vitro replication starting from the minimal ors8 origin, p186. Interestingly, both K36 and K58 anti-kin17 antibodies were able to reduce the replication activity of the p186 plasmid threefold compared to control levels (Fig. (Fig.6D).6D). This observation strongly suggested that kin17 protein is involved in earlier steps of DNA replication, presumably the initiation phase.

The prototype of the protein known to interact with origins and also engaged in both DNA replication and repair is Ku, the DNA binding subunit and the allosteric activator of the DNA-dependent protein kinase, DNA-PK (70). DNA-PK, which consists of Ku and the DNA-PK catalytic subunit (DNA-PKcs), is critical for end joining of DNA double-strand breaks and site-specific V(D)J recombination. Recent studies, using Ku knockout mice and yeast, support additional functions for Ku in transcription, telomeric maintenance, replicative senescence, and DNA replication (51, 60, 70). Ku is the DNA-dependent ATPase purified from HeLa cells (10), which cofractionated with a 21S multiprotein complex that is able to support SV40 DNA replication in vitro (71). Ku has been shown to bind to several origins of DNA replication (3, 18, 51, 60, 69) and to matrix attachment regions (24). These results suggested that Ku protein, if not already associated with the replication complex, will interact with other replication proteins and helps then in recruiting the other required proteins to the initiation site. A similar function could be proposed for kin17 protein. Another example is the Mre11 complex (composed of Mre11, Rad50, and Nbs1), required to activate a DNA damage-induced S-phase checkpoint in mammalian cells (54), that has been suggested to directly influence S-phase progression near replication origins via its interaction with the E2F1 transcription factor and at replication forks (40).

Various roles for the stress-activated nuclear kin17 protein have been proposed for important cellular metabolic processes, like repair, transcription, and replication. Our data strongly suggest a direct participation of kin17 protein in the DNA replication process by binding to origin DNA sequences. In order to shed some light on the link between replication factories at the origin of replication, additional experiments are needed to study the protein-protein interactions involved and the dynamic changes of kin17 protein after replication fork arrest induced by DNA-damaging agents.

Acknowledgments

This article is dedicated to the memory of Gerald B. Price (McGill Cancer Center, McGill University, Montreal, Canada).

We particularly thank Y. Frobert, C. Créminon, J. Grassi, and all the staff of the Service de Pharmacologie et d'Immunologie (DRM, CEA) for monoclonal antibodies against human kin17 protein; G. Baldacci and I. Tratner (Institut Curie, Orsay) for stimulating discussions, advice, and purified polyclonal antibody against PCNA; and E. Pichard for electron microscopy. B. Dutrillaux is gratefully acknowledged for his continuous support. We are grateful to S. Linn (Berkeley, CA) and A. M. Holmes (Waltham, MA) for providing DNA polymerase α antibody and B. Stillman for RFCp140 antibody.

Maria Zannis-Hadjopoulos, Olivia Novac, and Domenic Di Paola were supported by grants from the CIHR and FRSQ, respectively. This work was supported by Electricité de France (EDF) contract 8702.

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