Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation
© Tan et al; licensee BioMed Central Ltd. 2010
Received: 17 November 2009
Accepted: 16 February 2010
Published: 16 February 2010
Functional cooperation between FACT and the MCM helicase complex constitutes an integral step during DNA replication initiation. However, mode of regulation that underlies the proper functional interaction of FACT and MCM is poorly understood.
Methods & Results
Here we present evidence indicating that such interaction is coordinated with cell cycle progression and differential complex formation. We first demonstrate the existence of two distinct FACT-MCM subassemblies, FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Both complexes possess DNA unwinding activity and are subject to cell cycle-dependent enzymatic regulation. Interestingly, analysis of functional attributes further suggests that they act at distinct, and possibly sequential, steps during origin establishment and replication initiation. Moreover, we show that the phosphorylation profile of the FACT-associated MCM4 undergoes a cell cycle-dependent change, which is directly correlated with the catalytic activity of the FACT-MCM helicase complexes. Finally, at the quaternary structure level, physical interaction between FACT and MCM complexes is generally dependent on persistent cell cycle and further stabilized upon S phase entry. Cessation of mitotic cycle destabilizes the complex formation and likely leads to compromised coordination and activities.
Together, our results correlate FACT-MCM functionally and temporally with S phase and DNA replication. They further demonstrate that enzymatic activities intrinsically important for DNA replication are tightly controlled at various levels, thereby ensuring proper progression of, as well as exit from, the cell cycle and ultimately euploid gene balance.
Complete and precise DNA replication is essential to the maintenance of genomic integrity and balance. Initiation is the most critical regulatory step, which coincides with the onset of S phase and requires prior assembly of pre-replicative complexes (preRCs). Reinitiation of DNA replication is usually prevented, and only a single round of DNA duplication is performed in a cell cycle. Such restriction mechanism, called replication licensing, partly lies in the regulation of preRC assembly. The protein components of the preRC complex include origin recognition complex (ORC), Cdc6, Cdt1 and minichromosome maintenance proteins (MCM2-7). Phosphorylation of components of the assembled pre-RC constitutes a second level of initiation regulation, upon which the initiation of DNA replication is triggered at the G1-S boundary [1–3]. Finally, as with the formation of pre-RC, the transition to DNA replication involves the association of additional replication factors that facilitate unwinding of the origin DNA, as well as multiple DNA polymerases . Following origin activation, new DNA synthesis begins as replication forks move away from the initiation region [1, 5, 6].
Among different replication factors, the hexameric helicase complex MCM provides an essential activity, catalyzing the unwinding of DNA duplex . Previous work has established a direct role of MCM in not only the initiation step, but also the elongation stage of DNA replication [4, 8]. MCM possesses various functional features that are coordinated with other events of the cell cycle [1, 7]. Consistent with its functional significance, several regulatory mechanisms have been uncovered that serve to preserve and restrict its proper activities . Phosphorylation accounts for a major regulation. Activation of the MCM complex requires the actions of both the CDC7/DBF4 and cyclin-dependent kinases [1, 2]. Mitotic and DNA damage-induced phosphorylation of the MCM4 subunit, concomitant with loss of activity and/or subcellular localization change, involves CDK2-cyclin A or cyclin B [10–14]. Another mode of regulation lies in the combinatorial formation of MCM subassemblies. Aside from the expected heterohexameric complex (MCM2/3/4/5/6/7), in vitro experiments have demonstrated the formation of several stable subassemblies including MCM2/4/6/7, MCM4/6/7, and MCM3/5 complexes [15–18]. Among them, a weakly processive DNA helicase activity was identified in the MCM4/6/7 complexes of human, mouse, and fission yeast, whereas the heterohexamer lacks such activity [15, 16, 19, 20]. Work done by Schwacha and Bell further discriminated two functionally distinct MCM protein subgroups: the "catalytic core" MCM4/6/7 and the "regulatory" MCM2p, 3p, 5p . These results suggest that distinct assemblies of MCM subunits may contribute optimally to the coordinated and differential actions during the progression of replication.
Chromatin poses yet another type of regulation of the MCM activity, and the progression of replication in general, in an inhibitory fashion [1, 22]. Various reports have shown that local chromatin environment, as well as chromatin remodeling factors, directly dictates activity of the replication origin and DNA replication [23–28]. As demonstrated by our recent work, nucleosomes impose a structural hindrance that efficiently reduces the DNA helicase activity of MCM . Functional interaction between MCM and the FACT heterodimeric complex, however, alleviates such inhibition and concomitantly facilitates chromatin DNA unwinding. Our findings, together with those from other groups, show that the FACT-MCM complex plays an important role in the normal progression of DNA replication initiation and S phase in vivo [29–31]. Mode of regulation for this essential chromatin unwinding activity has not been characterized presently.
In this study we present several lines of evidence linking the functions of the FACT-MCM complex to cell cycle progression and regulation. Functional attributes such as origin association, DNA unwinding activities, and complex formation are intimately coordinated with cell cycle progression. Furthermore, the FACT-MCM interaction is generally dependent on persistent cell cycle and cell proliferation. These results suggest that the FACT-MCM complexes are tightly controlled at various levels to safeguard its essential activity as well as precise DNA synthesis.
Antibodies and Western blot analysis
Generation of monoclonal antibodies against SSRP1 (2B12/control, 10D1, and 10D7) and hSpt16p (8D2) was described previously . Anti-human ORC1 monoclonal antibody was purchased from NeoMarker/Lab Vision. Polyclonal serum against human pan-MCM was purchased from BD biosciences. Monoclonal antibody against Cdc6 and rabbit antisera against MCM3, MCM4, MCM5, and MCM6 were produced with the following peptide antigens and affinity purified by Dagene (Taiwan). Cdc6: MCM3: SDTEEEMPQVHTPKTAD; MCM4: SRRGRATPAQTPRSED; MCM5: KEVADEVTRPRPSGE; MCM6: KYLQLAEELIRPERNT. Polyclonal antibody against MCM2 was generated using a recombinant protein fragment of MCM2 (a.a. 792-892). Monoclonal antibody against Cdc45 (E-3) was purchased from Santa Cruz Biotechnology. Anti-FLAG mAb (M2) was purchased from Sigma. Western blot analysis was performed after electrophoretic separation of polypeptides by 7.5% or 10% SDS-PAGE and transfer to Hybond-C membranes. Blots were probed with the indicated primary and appropriate secondary antibodies, and detected by ECL chemiluminescence (Amersham).
HeLa or K562 cells were extracted using a buffer containing: 20 mM HEPES (pH 7.4), 0.2 M NaCl, 0.5% TX100, 5% glycerol, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 2 mM Na3VO4, 1 mM NaF, 1 mM DTT, plus protease inhibitors. For preparation of nuclear extracts, HeLa nuclei were isolated and lysed in nuclear extraction buffer (10 mM HEPES with pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% TX-100, 0.4 M NaCl, 10% glycerol and protease inhibitors). All immunoprecipitations were done with the indicated antibodies prebound to protein G-Sepharose (Amersham), and washed in the cell lysis buffer.
Gel filtration chromatography was done using a precalibrated Sephacryl S-400 HR column with a bed volume of 135 ml (Pharmacia). Nuclear lysate preparation, chromatographic settings, and fraction collection and processing were done essentially as reported previously .
Cell culture and cell cycle analysis
All HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin and streptomycin. Cells were transfected using Lipofectamine (GIBCO) according to the manufacturer's instructions. For monitoring cell cycle progression and immunostaining analysis, collection of HeLa cells at different stages of the cell cycle was achieved by the mitotic shake-off or the double thymidine block method, as outlined previously . Procedure for the FACS analysis was also described in the same report. Early G1 cells were collected by replating mitotic cells and allowing attachment for 2-4 hrs. To achieve a differentiation (or resting) phase in K562 cells, cells were subjected to 3-day treatment of 2 mM sodium butyrate .
To establish a plasmid-based dsRNAi system targeting endogenous MCM3 or MCM4, annealed oligonucleotides corresponding to partial sequence were designed and ligated to the pSuper.neo+GFP (OligoEngine) according to the manufacturer's instructions. The cDNA sequence of the targeted mRNA region for different genes is as follows. MCM3: 5'-AAACGAGAAGAGGGCTAAC-3' (nucleotides 171-189); MCM4: 5'-GACACCACACACAGTTATC-3' (nucleotides 1095-1113). The same sequence in the inverted orientation was used as the non-specific dsRNAi control.
Indirect immunofluorescence and confocal microscopy
All steps of the immunostaining procedure were essentially identical to a previous report . Subsequent to staining with the indicated primary antibodies, secondary antibody incubation was done for 1 h using Alexa 488 conjugated goat anti-mouse IgG and Alexa Fluor 594 conjugated goat anti-rabbit IgG (Moleular Probes, Inc). To visualize DNA, cells were counter-stained with DAPI. Stained cells were analyzed with the Zeiss LSM-510 inverted confocal laser-scanning microscope, using a 100X/NA 1.4 oil immersion or 40× objective lens.
DNA helicase assay
The substrate for the helicase assay was a partially heteroduplex DNA containing a 17-mer oligonuleotide (5'-GTTTTCCCAGTCACGAC-3') annealed to the M13mp18 (+) circular ssDNA (Amersham). Before annealing, the oligonuleotide was labeled at the 5' end with [γ-32P]ATP by polynucleotide kinase. The annealed substrate was subsequently purified on the MicroSpin G50 column (Amersham). MCM-containing FACT immunocomplexes were isolated by the indicated antibodies and from HeLa cells at the specified cell cycle stages. With the exception of the immobilized source of enzymatic activities, DNA helicase assay was performed essentially as described previously , with the addition of approximately 10-20 fmol of substrate. After deproteination, samples were resolved by electrophoresis (15% native PAGE/TBE) and autoradiographed.
Chromatin immunoprecipitation assays were modified from previously described methods . Briefly, HeLa cells (exponentially growing or synchronized) were cross-linked with 1% formaldehyde for 10 min at 37°C. The nuclei were isolated and sonicated into oligonucleosomes of ~500-600 bp in length. The sheared chromatin was immunoprecipitated overnight with protein G-agarose previously bound with the 10D1, 8D2, or control antibody. After extensive washes, the immunoprecipitates were subjected to deproteination and cross-linking reversal. The presence of genomic DNA in the precipitates was detected by PCR with the B48 primer set and a background primer set. The background primers anneal to a region with no annotated genes, 30 kb upstream of the lamine B2 origin sequence on chromosome 19, and have the following sequences: 5'-CTATGCCAAGCCCATTCTAGGTCCT-3 (sense); 5'-GCAGGGAAACTGTGCACAGCAAGAG-3' (antisense). Upon amplification for 27-30 cycles, the products were resolved by 2% agarose gels and visualized with ethidium bromide staining. UV-illuminated images were photographed and analyzed by AlphaImager 1220 (Alpha Innotech Corp.)
Subassemblies of MCM form distinct complexes with FACT heterodimer
The primary biochemical activity of the MCM complex is the unwinding of the DNA strands [15, 19]. Thus, to examine whether the identified MCM-associated FACT complexes are catalytically active, we performed DNA helicase assay. Immunoprecipitates were incubated with labeled substrate and their abilities to displace annealed oligonucleotide were assayed. We found that, as compared to the control antibody, FACT complexes precipitated by either 8D2 or 10D1 antibody possessed DNA helicase activity (Figure 1B). This suggests that the two distinct FACT immunocomplexes, although differing in MCM constituents, are both competent in unwinding DNA in vitro. Furthermore, such catalysis is an ATP-dependent process, as no such activity was detected in the absence of ATP, nor in the presence of nonhydrolyzable form of ATP (Figure 1C).
To further characterize whether the DNA helicase activity displayed by the FACT immunocomplexes is mediated through the associated MCM complex, we isolated from MCM4RNAi cells (Additional file 1 Figure S1B) the 10D1 immunoprecipitates that are deficient in the MCM complex and found that the DNA helicase activity was greatly reduced (Figure 1D). This reduction was not observed in the 10D1 immunoprecipitates isolated from control or MCM3RNAi cells (Additional file 1 S1B) in which the FACT-MCM association is still present. This result serves as strong evidence that the helicase activity of the 10D1 immunocomplexes can be attributed primarily to the associated MCM complex, but not any non-specifically associated activities.
Functional attributes of the two distinct FACT-MCM sub-complexes
To further evaluate the replicative role of these subcomplexes, we examined their origin association. In our previous work, we discovered that the 10D1-targeted FACT complex is present in vivo at a region of known replication origin, namely the replicator associated with the human lamin B2 gene. Using the ChIP assay, chromatin prepared from cells synchronized at different cell cycle stages was precipitated with the 8D2 or 10D1 antibody (Figure 3B). PCR reactions using specific sets of primers were subsequently performed to monitor the existence of the lamin B2 origin sequence (Figure 3B, lanes 1-9). As shown in Figure 3B, at equal loads of chromatin preparations, we observed specific occupancy of 8D2-targeted FACT complex in the ori region in asynchronously growing cells (compare lanes 4 and 7). As a control, sequence of a distant non-transcribed region (Figure 3B, lanes 10-12, see Methods) was not enriched in the immunoprecipitates. Furthermore, no origin binding of either immunocomplexes could be detected in mitotic cells (lanes 3 and 6, and ref. 28). This result is reminiscent of the observed dissociation of FACT from condensed chromatin during mitosis . Interestingly, while the 10D1-complex exhibited an enrichment of such origin binding during the transition from G1 to S phase (compare lanes 1 and 2; Figure 3C, histogram on the right), the 8D2-associated complex seemed to display a relatively greater degree of association in G1 as compared to the point entering S phase (lanes 4 and 5; Figure 3C, histogram on the left). These results reflect the activity of both FACT-MCM complexes locally at origins or replication forks, and imply their differential origin association.
Next, to further delineate the differential origin binding modes of the two FACT-MCM immunocomplexes, we performed additional ChIP assays on MCM3RNAi or MCM4RNAi cells. Chromatin preparations from these two cell lines were subjected to ChIP using either the 8D2 or 10D1 antibody and probed for the presence of the lamin B2 origin sequence (Figure 3D). In the context of downregulated MCM3 or MCM4 protein level, association of the 8D2 immunocomplex with the origin was disrupted (Figure 3D, middle panel). This signals that the origin binding of FACT (and the FACT-MCM2/3/4/5) lies, at least in part, in the presence of an intact MCM2/3/4/5 complex. Furthermore, in the MCM4RNAi cells, we saw a similar reduction of the origin fragment in the 10D1 immunocomplex, demonstrating the importance of MCM4 in this functional regard (top panel). Intriguingly, knockdown of the MCM3 subunit unexpectedly led to a weakened association of FACT-MCM2/4/6/7 with lamin B2 origin. A likely explanation for this phenomenon is that the origin association of the MCM3-associated complexes (including FACT-MCM2/3/4/5) may be preceding and required for the subsequent recruitment of FACT-MCM2/4/6/7. Therefore, there may exist two different temporal modes through which the FACT-MCM complexes are recruited to the replicator (see Discussion). Collectively, results presented in Figure 3 suggest that these two FACT-MCM complexes may operate at distinct stages during the initiation phase of DNA replication.
Functional regulation of the FACT-MCM complex mirrors cell cycle progression
Further evidence that links the possible role of FACT-MCM to S phase was obtained from immunofluorescence analysis. While examining the subcellular localizations of these two factors at different cell cycle stages, we found a discernible degree of colocalization between FACT and MCM4 at G1/S junction as well as throughout S phase (Figure 5B). However, this colocalization was markedly reduced in early G1 (Figure 5B) or G2/M cells (data not shown). Such manner of interaction between complexes may contribute to their coordinated functions during S phase.
Cell proliferation underlies the interaction between FACT and MCM
We made an interesting and novel finding of the existence of multiple assemblies of the FACT-MCM complex: FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Based on the previous observations on the stable formation of different subassemblies among the six MCM subunits [15–17, 21], we initially postulated that the identified FACT-MCM2/3/4/5 complex may serve as a regulator to the presumably "catalytic" assembly of FACT-MCM2/4/6/7. However, we subsequently detected DNA unwinding activities in both of the immunocomplexes. Despite this discrepancy, which may be attributed to the intrinsic difference between the endogenous (as in FACT-MCM) and the reconstituted complexes, the true architecture as well as the catalytic properties of the MCM complexes in the cellular context is still largely unknown. The existence of multiple DNA helicase subcomplexes (or the FACT-MCM subcomplexes) has several explanations. First, certain subcomplexes still may serve as regulators to other catalytic counterparts in the cell, as proposed previously. Such coordinated action potentially contributes to the highly regulated process of DNA duplication. Second, combinatorial association of subunits may serve as the underlying basis for differential origin selection (early vs. late). To this end, further functional elucidation of these complexes possibly will rely on better knowledge of mammalian origin organization and techniques such as high-resolution ChIP. Third, these complexes could differentially act at discrete steps during DNA synthesis (initiation vs. elongation, or early initiation vs. late initiation). Based on the data presented in Figure 3, which distinguish the pre-RC association and temporal mode of origin binding between the two subcomplexes, we favor this paradigm (Figure 7A). Our results are consistent with a model in which the FACT-MCM2/3/4/5 is first recruited to the origin at the stage of pre-RC formation and origin establishment. Its helicase activity may be responsible for local, albeit partial, chromatin unwinding. Upon pre-RC assembly and entry into S phase, FACT-MCM2/4/6/7 subsequently becomes associated with the origin region, either juxtaposed to the already bound FACT-MCM2/3/4/5 (Figure 7A, model I), or substituting its occupancy (model II). It may then be involved in unwinding the origin to a more global extent and facilitating the association of additional factors and establishment of replication fork. Importantly, our results provide a plausible explanation for the hitherto uncharacterized roles of multiple MCM subcomplexes. The dynamics of spatial and temporal distribution of these FACT-MCM subcomplexes is an important research subject in the future.
The cell cycle-dependent manner through which FACT and MCM physically and functionally interact with each other was demonstrated by immunostaining analysis (Figure 5B) and immunocomplex helicase assay (Figure 5A). The observed behaviors of the FACT-associated MCM4 recall the mitotic-specific hyperphosphorylation and functional downregulation of MCM4, as reported previously . Such mode of regulation, presumably through a conserved mechanism, suggests the involvement of cell cycle regulators such as Cdk2/cyclin A or cyclin B [10–13]. In addition to catalytic inactivation, mitotic hyperphosphorylation of MCM4 may concomitantly lead to dissociation from chromatin, as indicated by these reports. Additionally, in accordance with the finding by Ishimi and Komamura-Kohno , we observed a moderate but reproducible increase in catalytic activity of the FACT-MCM complex isolated from the G1/S-synchronized cells as compared to those at other phases (Figure 5A). Concurrently, there was a greater extent of mobility shift of MCM4 in the S phase immunocomplex as compared to that of asynchronous cells (lanes 1 and 2, Figure 4B). However, it is unclear at present whether or which signaling pathway underlies such modification and catalytic activation. Cdc7/Dbf4 (DDK) complex is a likely candidate kinase regulator . Identification of these regulators may be a future research subject, and understanding of this signaling pathway will aid in further characterization of FACT-MCM or MCM proteins in general. Taken together, the consequences of these phosphorylation events reflect the critical integration of replication with cell cycle as well as a temporal resetting of MCM activity.
We thank Hiroshi Nojima for the MCM plasmids and Brian Calvi for the fly strains. We are especially grateful to the members of the S.C. Lee and C.T. Chien labs for technical assistance. This work was funded by National Science Council (NSC 98-2321-B-002-001 to SCL; NSC 97-2320-B-182-027-MY3 to BCMT), Institute of Biological Chemistry, Academia Sinica (to SCL), and Chang Gung Memorial Hospital (CMRPD160193 and CMRPD170302 to BCMT)
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