Article |
Address correspondence to Philippe Collas, Institute of Medical Biochemistry, P.O. Box 1112 Blindern, University of Oslo, Oslo 0317, Norway. Tel.: 47-2285-1066. Fax: 47-2285-1058. E-mail: philippe.collas{at}basalmed.uio.no
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Abstract |
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Key Words: chromosome; lamina-associated polypeptide; HA95; nuclear envelope; replication
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Introduction |
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We, and others, have cloned a 95-kD nuclear protein named HA95/NAKAP95/HAP95 (Orstavik et al., 2000; Seki et al., 2000; Westberg et al., 2000). HA95 displays partial homology to AKAP95, a member of the A-kinase anchoring protein family, but it lacks a protein kinase A (PKA)binding domain. HA95 appears in photobleaching experiments as a stable protein and cofractionates with chromatin and a nuclease- and salt-resistant "matrix." HA95 associates with itself (Orstavik et al., 2000) and interacts with RNA helicase A (RHA) to enhance expression of a constitutive transport element (CTE) involved in nuclear export of retroviral RNA (Westberg et al., 2000; Yang et al., 2001). In vitro nuclear breakdown and reassembly assays combined with antibody-blocking experiments have shown that HA95 is involved in NE breakdown and chromatin condensation, whereas a role in nuclear membrane reassembly is unlikely (Martins et al., 2000).
Initiation of DNA replication involves the assembly of prereplication complexes (preRCs) at origins of replication in G1 (Kelly and Brown, 2000; Bell and Dutta, 2002). PreRCs include the origin recognition complex (ORC), the minichromosome maintenance (MCM) complex, and the monomeric Cdc6 protein (Bell and Dutta, 2002). ORC recruits Cdc6 into the preRC in G1, and Cdc6 in turn promotes loading of MCM proteins on chromatin (Bell and Dutta, 2002). In human cells, Cdc6 levels are relatively stable during interphase (Williams et al., 1997; Saha et al., 1998). Nonetheless, a fraction of Cdc6 is exported out of the nucleus (Coleman et al., 1996) while another remains associated with chromatin during S and G2 phases (Coverley et al., 2000; Mendez and Stillman, 2000). Proteolysis of free Cdc6, not assembled into preRCs, has also been reported (Coverley et al., 2000). Thus, after origin firing at the start of S phase, preRCs are dissociated, ensuring a single round of replication per cell cycle.
We provide evidence here that HA95 interacts with LAP2ß via two distinct domains. Blocking the association of HA95 with LAP2ß does not affect nuclear assembly in vitro. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2ß triggers proteasome-mediated degradation of Cdc6 and inhibits initiation, but not elongation, of replication. The results suggest an implication of the association of HA95 with LAP2ß in regulating the initiation phase of DNA replication.
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Results |
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Mapping of the HA95-binding domains of LAP2ß
To map the domains of LAP2ß involved in the interaction with HA95, GSTLAP2ß fusion polypeptides were produced (Fig. 2 A) (Furukawa et al., 1995, 1997, 1998). Binding of each peptide to HA95Myc was determined in GST precipitations after incubation of the peptides in a nuclear extract from Bjab cells expressing HA95Myc. Control extracts were incubated with GST or glutathione beads alone. GST precipitates were immunoblotted using anti-Myc antibodies. Fig. 2 B shows that LAP2ß(1452), (1397), (1298), (137373), (137298), (243397), (243373), (299397), and (299373) precipitated HA95Myc. In contrast, LAP2ß(1193), (185), or (194298) did not precipitate HA95Myc. Thus, a first HA95-binding domain localizes to amino acids 137242 of LAP2ß and a second domain coincides with the lamin Bbinding domain at residues 299373. We designated these domains HA95-NBD (for HA95 NH2-terminal binding domain) and HA95-CBD (HA95 COOH-terminal binding domain), respectively.
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Direct association of LA2ß with HA95 was demonstrated in an overlay assay. HA95-IP proteins were resolved by SDS-PAGE, blotted, and overlaid with 50 µM of each GSTLAP2ß fragment. The same GSTLAP2ß peptides found to precipitate HA95 also bound HA95 in the overlay, as detected with anti-GST antibodies (Fig. 2 D, top). Moreover, all LAP2ß fragments containing the lamin Bbinding domain (residues 299373) bound to immunoprecipitated and immobilized lamin B (Fig. 2 D, bottom). However, two of the HA95-binding peptides, LAP2ß(1298) and LAP2ß(137298), did not bind lamin B, confirming the existence of an HA95-binding domain (HA95-NBD) distinct from the lamin Bbinding region.
To determine whether HA95-binding LAP2ß peptides would disrupt endogenous HA95LAP2ß association, dissociation experiments were performed using GSTLAP2ß peptides harboring both HA95-binding domains (LAP2ß [137373]) or either HA95-NBD (LAP2ß[137298]) or HA95-CBD (LAP2ß[299373]). HA95-IPs were incubated for 1 h with 100 µM GSTLAP2ß peptides, HA95-IPs were sedimented, and dissociation of LAP2ß from HA95 was monitored by immunoblotting. LAP2ß(137373) completely dissociated LAP2ß from HA95-IP (Fig. 2 E, lanes 1 and 2), however peptides containing HA95-NBD or HA95-CBD were ineffective (lanes 36). Nevertheless, two sequential 30-min incubations of HA95-IPs with LAP2ß(137298) followed by LAP2ß(299373) led to dissociation of the complex (lanes 7 and 8), and reversing the order of peptide addition produced similar results (lanes 9 and 10). We concluded that disruption of both HA95-binding domains was required for dissociation of LAP2ß from HA95 in vitro. The data also argue that LAP2ß(137298) and LAP2ß(299373) peptides are capable of dissociating HA95 from HA95-NBD and HA95-CBD, respectively.
Inhibition of LAP2ß binding to HA95 does not affect nuclear reassembly in vitro
To determine whether interaction between HA95 and LAP2ß was involved in NE assembly, we assessed the ability of HA95-binding GSTLAP2ß peptides to compete with LAP2ß for membrane targeting to chromosomes. Purified HeLa nuclei were disassembled in mitotic extract. The resulting condensed chromosomes contained HA95 and BAF, but no A- or B-type lamins, LAP2ß, or lamin B receptor (LBR) (Fig. 3 A). After preincubation with 10 µM of each GSTLAP2ß peptide, chromosomes were sedimented and peptide binding was examined by immunofluorescence using anti-GST antibodies. All peptides except GSTLAP2ß(194298) bound chromatin (Fig. 3 B), as anticipated from their ability to bind HA95 (Fig. 2), DNA, or chromatin (see Introduction). Chromosomes were resuspended in interphase extract (Fig. 3 C, Input chrom.) under conditions promoting nuclear assembly. Nuclear morphology was examined by phase contrast microscopy and membrane labeling with DiOC6 after 2 h (Fig. 3 C). Without peptide or with GST alone, >80% of chromatin masses supported nuclear reformation. In contrast, LAP2ß fragments (1452), (1397), (137373), (243397), (299397), (243373), and (299373) inhibited nuclear assembly. Each of these peptides contained the lamin Bbinding domain/HA95-CBD. Peptides that do not bind HA95 (nor lamin B) (LAP2ß[1193], [185], and [194298]) did not block membrane assembly, neither did LAP2ß(1298) or (137298), which both contain HA95-NBD.
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LAP2ß(137298) inhibits initiation of DNA replication in intact nuclei in vitro
To determine whether interphase nuclear functions were affected by the HA95LAP2ß association, we monitored the effect of disrupting the LAP2ßHA95 interaction on DNA replication in purified G1-phase nuclei. To this end, we adapted an in vitro replication assay from that of Krude et al. (1997).
Nuclei were isolated from G1-phase HeLa cells. GSTLAP2ß peptides were introduced into the nuclei after mild treatment with lysolecithin. Lysolecithin was previously shown not to affect dynamic properties of isolated nuclei in in vitro nuclear disassembly assays (Collas et al., 1999). Peptides were taken up by 90% of the nuclei, as shown by immunofluorescence using anti-GST antibodies (Fig. 4 A; GSTLAP2ß[1452] is shown). Control and peptide-loaded nuclei were incubated for 3 h in a concentrated (2530 mg/ml) nuclear and cytosolic extract from S-phase HeLa cells containing [
32P]dCTP, dNTPs, GTP, and an ATP-regenerating system to promote replication. Under these conditions, G1 nuclei loaded with GSTLAP2ß(1452) were capable of importing an exogenous BSAnuclear localization signal conjugate (unpublished data) or the replication factor Cdc6 (Fig. 4 B). Import was ATP and GTP dependent and blocked by preincubation of the nuclei with antibodies against nucleoporins (Fig. 5 B, mAb414). These results indicate that import took place through nuclear pores rather than passively through a damaged NE, and confirmed a previous report of physiological import of transcription factors by nuclei purified as previously described (Landsverk et al., 2002). We also tested whether peptides containing HA95-NBD or HA95-CBD introduced into G1 nuclei would inhibit nuclear import under the conditions described above, as this would be expected to affect DNA replication. Fig. 4 C shows that none of the peptides distinctly impaired import of Cdc6. Notably, import was permitted by the HA95-NBDcontaining peptide LAP2ß(137298) and blocked by mAb414. Lastly, the nuclear DNA did not undergo any detectable degradation upon incubation of the G1 nuclei in the extract at 4°C or 37°C, as judged by TUNEL analysis (Fig. 4 C) and DNA agarose gel electrophoresis (Fig. 4 D). These results indicate that isolated G1 nuclei are functional in import, can be manipulated to introduce peptides, and do not undergo detectable DNA degradation in S-phase extract, and that LAP2ß fragments containing either HA95-binding domain do not block nuclear import of Cdc6 in vitro.
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Initiation of replication requires the cyclin ACdk2 complex (Stoeber et al., 1998). Thus, to provide evidence that DNA synthesis in G1 nuclei was due to true initiation of replication, we showed that DNA synthesis was inhibited with 10 µM of the Cdk2 inhibitor, olomoucine, in the assay (Stoeber et al., 1998) (Fig. 5 B). Similar results were obtained with 500 µg/ml dimethylaminopurine, a nonspecific protein kinase inhibitor (unpublished data). Altogether, the results indicate that peptides containing HA95-CBD partially inhibit replication in G1 nuclei. However, those containing HA95-NDB completely abolish replication. We ruled out an involvement of the GCL-binding domain of LAP2ß (residues 219298) in inhibition of replication by fragment 137298 because LAP2ß(194298) was not inhibitory.
As described earlier in the nuclear reconstitution experiment, none of the peptides altered the immunofluorescence labeling pattern of LAP2ß, A- and B-type lamins, or BAF in the G1 nuclei examined (unpublished data; see also below). In addition, Western blot analysis of the G1 nuclei loaded with the peptides shows that neither peptide affected BAF levels (or HA95) in the nuclei (Fig. 5 C). As LAP2ß(1193) and (137298) block replication whereas LAP2ß(194298) and (185) allow replication (Fig. 5 D), the data suggest that region 137193 of LAP2ß is involved in replication initiation without affecting the distribution of NE proteins or the amount of BAF in the nuclei.
To establish that the LAP2ß(137298) peptide inhibited semiconservative DNA replication in G1 nuclei, BrdU substitution and density gradient centrifugation of 32P-labeled DNA was performed. G1 nuclei loaded with no peptide, GST alone, or LAP2ß(137298) were incubated for 3 h in S-phase extract containing BrdU and [32P]dCTP to quantitate replication. DNA was analyzed by CsCl gradient centrifugation. In control nuclei, the cytosol promoted DNA synthesis, producing primarily hemisubstituted (heavy-light [HL]) DNA, indicative of semiconservative replication (Fig. 5 E). In contrast, no peak of hemisubstituted DNA was detected in nuclei loaded with LAP2ß(137298), consistent with replication inhibition detected by 32P incorporation. We concluded that LAP2ß(137298) interfered with semiconservative replication in G1 nuclei.
Disrupting LAP2ßHA95 interaction does not affect the elongation phase of DNA replication in vitro
We next determined whether the elongation phase of replication was also affected by LAP2ß peptides. Nuclei isolated from S-phase HeLa cells were loaded with each GSTLAP2ß peptide (Fig. 5 F, left; GSTLAP2ß[1452] is shown) and incubated in S-phase extract under conditions promoting replication. Remarkably, all peptides supported DNA synthesis to the same extent as a control without peptide (Fig. 5 F). In addition, to validate our DNA replication assay, we showed that DNA synthesis occurred in S-phase nuclei incubated in extract from G0 cells, reflecting the replicating state of these nuclei (Fig. 5 B). In contrast, G0 nuclei did not synthesize DNA in S-phase extract. Note, however, a faint 32P label in G0 nuclei in the S-phase extract, due to a minor proportion of slowly replicating nuclei in the G0 cell population (unpublished data).
HA95 coimmunoprecipitates with the Cdc6 protein in G1 phase
Initiation of DNA replication requires the assembly of preRCs at origins of replication in G1. The chromatin-bound ORC complex recruits Cdc6, which in turn promotes targeting of MCM proteins. HA95 and Cdc6 were found to coimmunoprecipitate from G1-phase HeLa cells; nevertheless, whereas anti-HA95 antibodies precipitated all detectable Cdc6, a substantial fraction of HA95 did not associate with the Cdc6 immune precipitate (Fig. 6 A). In S phase however, Cdc6 and HA95 did not coprecipitate (Fig. 6 A), despite the reported persistence of a fraction of Cdc6 on chromatin beyond G1 (Mendez and Stillman, 2000).
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The ORC large subunit has recently been shown to be degraded by ubiquitin-mediated proteolysis in human cells (Mendez et al., 2002). Thus degradation of Cdc6 by the 25S proteasome was examined. Inhibition of the proteasome by incubation of nuclei containing LAP2ß(137298) with 25 µM of the proteasome inhibitors LLnL or ß-lactone during peptide loading and incubation in buffer prevented degradation of Cdc6 (Fig. 6 B, lanes 6 and 7). However, the calpain inhibitor LLM, used as a control, did not block Cdc6 proteolysis (lane 8). Inhibition of degradation of p53, a known substrate of ubiquitin-mediated proteolysis, with LLnL or ß-lactone confirmed the efficacy of the inhibitors (Fig. 6 B). Introduction of LAP2ß(137298) in S-phase nuclei did not elicit degradation of Cdc6 (or p53 or Orc2; unpublished data). These results suggest that dissociation of HA95 from HA95-NBD in G1- but not S-phase nuclei triggers degradation of Cdc6 by the proteasome. Because p53 is also proteolyzed by LAP2ß(137298), the data also suggest that the peptide promotes degradation of a subset of nuclear proteins.
As G1 nuclei containing LAP2ß(137298) do not replicate DNA in S-phase extract, we determined whether proteasome inhibitors would rescue S-phase entry of these nuclei. G1 nuclei were loaded with LAP2ß(137298) in the presence of LLnL or ß-lactone and incubated for 3 h in S-phase extract containing [32P]dCTP under conditions promoting replication. Fig. 6 C shows that LLnL and ß-lactone relieved the inhibition of DNA replication imposed by LAP2ß(137298). The calpain inhibitor LLM, however, had no effect and, as expected, LLnL or ß-lactone did not affect replication of LAP2ß(299373)-loaded G1 nuclei (Fig. 6 C). Therefore, inhibition of the proteasome enables entry of the G1 nuclei into a replication phase.
Injection of LAP2ß(137298) into G1 nuclei inhibits entry into S phase in vivo
The significance of HA95 interaction with HA95-NBD or HA95-CBD was further investigated in vivo by injections of 5 nM GSTLAP2ß peptides into the nuclei of HeLa cells in early G1 (2 h after release from mitotic arrest). Injections were verified by nuclear retention of a 150-kD FITCdextran (see below and Fig. 8 A).
We first assessed whether the distribution of INM and lamina proteins was altered in the injected nuclei. As seen earlier in in vitroreconstituted nuclei, GSTLAP2ß peptides were detected throughout the nucleus for the most part, with a propensity of the anti-GST antibody to decorate the nuclear periphery more strongly (Fig. 7, GST). This, however, was not specific for the peptide injected (Fig. 7, bottom three rows). Immunofluorescence analysis of peptide- and mock (buffer)-injected cells indicated that LAP2ß and B-type lamins remained localized at the NE 23 h after injection with either peptide (Fig. 7). Similar results were obtained for LBR, emerin, and A-type lamins (unpublished data). Additionally, no alteration in the localization of BAF in peptide-injected and control cells was detected (Fig. 7). BAF remained distributed throughout the nucleoplasm with an enrichment around the periphery. Thus, we could not attribute a noticeable effect of intranuclear peptide injection in G1 on overall nuclear architecture.
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Discussion |
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Interaction between HA95 and LAP2ß is dispensable for nuclear membrane assembly
We previously reported that anti-HA95 antibody-mediated blocking of HA95 function on condensed chromosomes had no inhibitory effect on nuclear membrane assembly in vitro (Martins et al., 2000). This suggested that HA95 may not be involved in membrane targeting to chromatin. Our results indicate that any LAP2ß fragment that can bind lamin B inhibits nuclear assembly, however peptides that bind HA95, but not lamin B, fail to inhibit assembly. Thus, competition for LAP2ß binding to HA95 has no detectable effect on nuclear formation. Although our results show that the lamin Bbinding domain of LAP2ß is involved in NE formation (Yang et al., 1997; Gant et al., 1999), they document a lack of effect of HA95 binding activity on nuclear assembly.
Role of association of HA95 with the NH2-terminal HA95-binding domain of LAP2ß in DNA replication
Our results suggest that the HA95LAP2ß interaction via HA95-NBD may represent an additional form of control for replication initiation, at least in transformed cells. We ruled out the possibility that disruption of the HA95LAP2ß interaction upon intranuclear introduction of LAP2ß fragments affected overall nuclear organization, which in itself might compromise DNA replication (Gant et al., 1999; Moir et al., 2000). Notably, the localization of endogenous LAP2ß, A- and B-type lamins, and BAF does not seem to be perturbed by disruption of the interaction of HA95 with HA95-NBD upon nuclear reassembly in vitro, in isolated G1 nuclei, or in G1-phase nuclei in vivo. Additionally, BAF levels remain unaffected in G1 nuclei, arguing that defects in replication induced by LAP2ß disruptor peptides do not result from changes in BAF protein levels (Segura-Totten et al., 2002). The HA95-NBD of LAP2(ß) partially overlaps with the LEM domain, which interacts with BAF. BAF distribution and levels are not altered by LAP2ß peptides containing HA95-NBD, and regions nonoverlapping with the HA95-binding domain were sufficient for BAF binding in yeast two-hybrid assays (Furukawa, 1999). Nonetheless, whether manipulating the association of HA95 with LAP2ß interferes with binding of LAP2 proteins to BAF remains to be examined (Shumaker et al., 2001).
How does the HA95LAP2ß interaction affect DNA replication? First, we should emphasize that the domain of HA95-NBD involved in replication (residues 137193; Fig. 9) is almost entirely included in the 187 amino acids (1187) common to all LAP2 proteins. Thus, the HA95LAP2 interaction is probably not restricted to the NE. We propose a hypothesis whereby interaction of HA95 with the NH2-terminal domain of LAP2 proteins and with components of the preRC brings components of the preRC to replication origins, maintains integrity of the preRC, and/or protects preRC components from degradation. It is conceivable that destabilizing the HA95LAP2ß interaction in G1 may displace preRC components, triggering their proteasome-mediated degradation and, as a result, blocking replication initiation. These alternatives are compatible with the distribution of preRCs throughout the genome and the intranuclear localization of LAP2, a nonmembrane-bound LAP2 isoform (Vlcek et al., 1999). Disruption of the interaction specifically abolishes S-phase entry but has no effect on the elongation phase of replication. Furthermore, HA95 and Cdc6 coimmunoprecipitate in G1 but not S phase, suggesting that HA95 (in)directly interacts with the preRC. The lack of interaction of HA95 with Cdc6 in S phase is consistent with nuclear export or degradation of a fraction of Cdc6 in mammalian cells (Saha et al., 1998; Coverley et al., 2000) and with disassembly of the preRC.
Disruption of HA95 interaction with HA95-NBD in G1 triggers proteasome-mediated degradation of Cdc6 (and p53), whereas proteasome inhibitors block LAP2ß(137298)-induced proteolysis and rescue the replication inhibitory effect of the LAP2ß peptide in G1 nuclei. Yet, the inhibitors obviously did not rescue the LAP2ßHA95 interaction, indicating that this association is dispensable for replication. It is likely that the LAP2ß(137298) peptide causes degradation of a class of nuclear proteins, because p53 reacts similarly to Cdc6. This could result from a pleiotropic effect of the peptide, perhaps disturbing other proteins that interact through a similar domain. In any event, inhibition of DNA synthesis by LAP2ß(137298) may be indirect, for example by activating a checkpoint pathway. This speculative hypothesis remains to be tested.
Our results extend the notion that the NE contributes to regulating replication. In Xenopus, disruption of lamina organization alters the distribution of replication factors and inhibits the elongation phase of DNA synthesis (Moir et al., 2000). Thus, replication may be regulated at several levels by nuclear lamins and nuclear membranechromatin interactions, in addition to interactions between chromatin-associated proteins.
Multiple functions of HA95 dictated by its interaction with multiple ligands
By interacting with several intranuclear ligands, HA95 emerges as a multifunctional molecule. In addition to its involvement in replication, HA95 binds RHA (Westberg et al., 2000), suggestive of a role in transcription regulation. RHA binds to a CTE involved in nuclear export of unspliced viral RNA, and association of HA95 with RHA enhances CTE-mediated gene expression and promotes nuclear export of unspliced mRNA (Yang et al., 2001). By binding to LAP2ß, HA95 may favor viral RNA export by tethering it near the NE.
HA95 also interacts with the catalytic subunit of PKA within the nucleus and localizes the catalytic subunit to sites where it can modulate transcription from specific promoters (Han et al., 2002). Thus, HA95 may act as a targeting molecule for the PKA catalytic subunit in the nucleus. Along this line, it is tempting to speculate that HA95LAP2ß, or HA95LAP2, interactions may promote targeting or stabilization of signaling molecules in the vicinity of transcription sites and at origins of replication at discrete stages of the cell cycle.
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Materials and methods |
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Cells and nuclei
Bjab cells were grown in RPMI 1640/10% FCS (GIBCO BRL) (Orstavik et al., 2000). HeLa cells were cultured in EMEM/10% FCS (GIBCO BRL). HeLa cells were synchronized in M phase with 1 µM nocodazole for 18 h. To allow cell cycle reentry, cells were plated at 2.5 x 106 cells in 162-cm2 flasks. G1- and S-phase cells were harvested (or microinjected) 2 and 12 h, respectively, after release from mitotic arrest. Cells were arrested in G0 by a 5-d culture under confluent conditions without serum. Nuclei were isolated from Bjab cells and from G0-, G1-, or S-phase HeLa cells by Dounce homogenization (Martins et al., 2000). For nuclear reassembly assays, nuclei were isolated from confluent HeLa cells and used fresh of frozen/thawed (Steen et al., 2000). Freshly isolated nuclei were used in replication assays.
Microinjection and BrdU incorporation
HeLa cells released from M-phase arrest were seeded on coverslips. Within 2 h, nuclei were microinjected with 25 pl PBS containing 10 µg/ml 150-kD FITCdextran to visualize injections, and 5 nM indicated GSTLAP2ß peptide. Cells were cultured in EMEM/10% FCS for up to 10 h without or with 100 µM BrdU and processed for immunofluorescence or BrdU incorporation analysis. S-phase cells were injected 1012 h after release from M-phase arrest. Approximately 50 cells were injected per treatment in two to three replicates. To detect BrdU incorporation, cells were fixed with methanol for 5 min, blocked, and overlaid with anti-BrdU antibodies (1:200 dilution) and TRITC-conjugated anti-BrdU antibodies.
Immunological procedures
Immunoblotting analysis was performed as previously described (Martins et al., 2000) using antibodies against HA95 (1:250 dilution), Myc (1:1,000), GST (1:1,000), B-type lamins (1:1,000), lamin A/C (1:500), LAP2ß (1:500), LBR (1:500), BAF (1:500), p53 (1:500), Cdc6 (1:500), and Orc2 (1:500). For immunoprecipitations, cells or nuclei were sonicated in IP buffer (10 mM Hepes, pH 7.5, 10 mM KCl, 2 mM EDTA, 1% Triton X-100, 1 mM DTT, and protease inhibitors) and lysates were centrifuged at 15,000 g for 15 min. Immunoprecipitations were performed from the supernatants with relevant antibodies (1:50 dilutions) as described earlier (Martins et al., 2000).
GST precipitation
Nuclei isolated from Bjab cells (109 nuclei/ml) were sonicated in GST precipitation buffer (300 mM KCl, 20 mM Hepes, pH 7.6, 0.1% Triton X-100, 1 mM DTT, 5 mM benzamidine, and protease inhibitors) and the lysate was centrifuged at 10,000 g. The supernatant (nuclear extract) was incubated with specified GSTLAP2ß peptides (1 or 5 µg/µl) overnight at 4°C with rotation. GST precipitations were performed using glutathione beads coated with 10 mg/ml BSA and pellets were washed in GST precipitation buffer. Proteins were eluted in SDS sample buffer.
Overlay assays
HA95-IPs proteins resolved by 10% SDS-PAGE were blotted onto nitrocellulose, blocked with 5% milk in Tris-buffered saline/0.01% Tween 20 (TBST) for 1 h, washed in TBST, and overlaid with 10 µM GSTLAP2ß peptides for 2 h in TBST. Membranes were washed in TBST and peptide binding was detected using anti-GST antibodies and peroxidase-conjugated secondary antibodies.
Nuclear reconstitution assay
Condensed, membrane-free chromatin masses were prepared from HeLa nuclei disassembled in mitotic extract (Martins et al., 2000). After sedimentation through 1 M sucrose, chromosomes were resuspended in peptide-binding buffer (100 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 20 mM Hepes, pH 7.5, 1 mM DTT, and protease inhibitors) containing 5 µM of indicated GSTLAP2ß peptides.
After 1 h at room temperature, chromosomes were sedimented, washed, and either immunologically analyzed or resuspended in nuclear reassembly extract. In this case, chromosomes (2 µl) were resuspended in 40 µl of an interphase HeLa cell 200,000 g cytosolic extract at 5,000 chromatin masses/µl, containing 4 µl mitotic membranes, an ATP-regenerating system (1.2 µl), and 100 µM GTP (0.4 µl) (Steen et al., 2000). After 2 h at 30°C, nuclear assembly was examined by phase contrast microscopy, membrane staining with 10 µg/ml DiOC6, or by immunofluorescence. Nuclei or chromatin masses were also sedimented through 1 M sucrose, washed, and solubilized in SDS sample buffer.
Loading of nuclei with GSTLAP2ß peptides
Peptide loading into isolated HeLa nuclei was performed as described earlier (Collas et al., 1999). In brief, nuclei were mildly permeabilized with lysolecithin and incubated for 1 h with indicated GSTLAP2ß peptides (100 µM) or GST alone. Nuclei were washed by sedimentation through 1 M sucrose and held on ice until use. Lysolecithin-treated nuclei were capable of active protein import in vitro (see Results).
In vitro replication and quantification of DNA synthesis
Replication was assayed by incorporation of [32P]dCTP into newly synthesized DNA. Isolated G0-, G1-, or S-phase nuclei (preloaded with GSTLAP2ß peptides, as indicated) were incubated for 3 h at 5,000 nuclei/µl in 40 µl of S-phase extract (unless indicated otherwise) containing the ATP-regenerating system, 100 µM GTP, a buffered mix of dNTPs (40 mM Hepes, pH 7.8, 7 mM MgCl2, 0.1 mM each of dATP, dGTP, dTTP, and dCTP; 2 µl) and 1 µl [
32P]dCTP (3,000 Ci/mmol; Amersham Biosciences). Concentrated S-phase whole cell extracts were prepared from S-phase HeLa cells collected 15 h after release from mitotic arrest. Cells were lysed by Dounce homogenization in cell lysis buffer (Martins et al., 2000), and then briefly sonicated on ice to lyse nuclei and release soluble nuclear components. The lysate was sedimented at 15,000 g for 15 min and then at 200,000 g for 2 h, both at 4°C. Protein concentration of the extract was 2530 mg/ml.
At the end of incubation in extract, samples were mixed with 1 volume of 20 mM Tris (pH 7.5) and 1 mg/ml proteinase K and digested for 2 h at 37°C. Samples were mixed by pipetting and 5-µl aliquots were electrophoresed through 0.8% agarose. Gel loading was assessed by ethidium bromide staining. Samples contained equal numbers of nuclei and sedimentation steps were eliminated to avoid loss of nuclei (Gant et al., 1999). Signals were quantified by phosphorImaging or by autoradiography.
BrdU density substitution
Density substitutions were done as previously described (Gant et al., 1999) in reactions consisting of isolated G1 nuclei in S-phase extract containing [32P]dCTP and 0.5 mM BrdU. Samples were digested with proteinase K and DNA was extracted with phenolchloroform then chloroform, ethanol precipitated, and dissolved in 100 µl Tris-EDTA buffer. Samples were mixed with 12 ml of 1.75 g/ml CsCl and centrifuged for 45 h at 60,000 g. 40 fractions were collected, an aliquot was counted by liquid scintillation, and the refractive index of each fraction was measured (Gant et al., 1999).
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Footnotes |
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Acknowledgments |
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This work was supported by the Portuguese Foundation for Science and Technology (S. Martins), the Research Council of Norway, the Norwegian Cancer Society, and the Human Frontiers Science Program (P. Collas).
Submitted: 7 October 2002
Revised: 9 December 2002
Accepted: 9 December 2002
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