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Molecular and Cellular Biology, June 2004, p. 4710-4719, Vol. 24, No. 11
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.11.4710-4719.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Institute of Veterinary Biochemistry and Molecular Biology,1 Institute of Molecular Biology, University of Zürich, CH-8057 Zürich, Switzerland,3 Cancer Research UK, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom2
Received 22 December 2003/ Returned for modification 20 January 2004/ Accepted 27 February 2004
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ABSTRACT |
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INTRODUCTION |
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Recently, much attention has been paid to the characterization of proteins that associate with the RecQ family of DNA helicases in order to understand their exact roles in the maintenance of genome stability in human cells (19, 34). BLM has been reported to interact with several proteins, including replication protein A (RPA), a DNA binding protein that plays essential roles during DNA replication, repair, and recombination (8); topoisomerase III
, a protein that, together with BLM, can resolve intermediates that arise during homologous recombinational repair (53, 54); BRCA1, a protein required for efficient homologous recombination and which acts as a signal processor for DNA damage responses (50); RAD51 and RAD51L3, proteins required for DNA strand invasion and exchange during homologous recombination (6, 7, 52); p53, a tumor suppressor implicated in S-phase processes, DNA repair, and homologous recombination (11, 41, 49); TRF2, a telomere binding protein (36, 44); WRN, Werner syndrome protein, another member of the RecQ helicase family (48); ATM, one of the essential transducers of DNA damage checkpoint responses (5); MLH1 and MSH6, proteins involved in mismatch repair and recombination (26, 37, 38); and five Fanconi anemia proteins: FANCA, FANCG, FANCC, FANCE, and FANCF, a multiprotein complex suggested to function in a DNA damage response pathway (31). The phenotypes exhibited by BS cells, as well as the proteins that can associate with BLM protein, imply that BLM may be involved in DNA recombination and repair processes linked to DNA replication and in telomere maintenance. There are several lines of evidence to show the importance of BLM during DNA replication. First, in Xenopus egg extracts, depletion of the Xenopus BLM homolog (xBLM) leads to an inhibition of DNA replication that can be rescued by addition of recombinant xBLM (27). Second, BLM–/– cells possess replication defects that include the accumulation of abnormal replication fork intermediates, retarded replication fork progression, and the delayed maturation of Okazaki fragments (10). Third, BLM is localized to replication foci, particularly during late S phase (51). However, it remains unclear whether the involvement of BLM in DNA replication is direct or is a secondary effect of lacking efficient repair of stalled replication forks. It is therefore of considerable interest to study the mechanism of how BLM influences DNA synthesis.
In this paper, we report that BLM physically and functionally associates with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1). CAF-1 is a three-subunit complex that promotes the incorporation of histones H3 and H4 into newly synthesized DNA during replication (47) or during nucleotide excision repair (15). Moreover, we show that BLM and hp150 colocalize to sites of DNA synthesis when cells are exposed to DNA replication inhibitors or DNA-damaging agents. In pursuit of a biological function of the BLM-hp150 interaction, we found that BLM specifically attenuates CAF-1-mediated chromatin assembly during DNA repair in vitro. Furthermore, we show that in BLM–/– cells, CAF-1 activity is not altered but that the absence of BLM affects the localization of CAF-1 following hydroxyurea (HU) treatment. Collectively, our data indicate that BLM and CAF-1 may function together in vivo during DNA metabolic processes and provide a novel insight into the role of BLM and CAF-1 in the maintenance of genome stability.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro binding assay. Different amounts (1.25, 2.5, and 5 pmol) of purified recombinant BLM, which was diluted in protein storage buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 1 mM ß-mercaptoethanol, 25% [vol/vol] glycerol) were dotted on a nitrocellulose membrane (MSI NitroBind); bovine serum albumin (BSA) and UvrD were dotted as control proteins. After being air dried for 15 min, the membrane was blocked for 2 h at room temperature in Tris-buffered saline (TBS) supplemented with 2.5% (wt/vol) milk and 0.05% (vol/vol) Tween 20 and then incubated for 3 h at 4°C, with different proteins that were 35S labeled using the TNT quick transcription-translation system (Promega), in 1 ml of TBS with 0.1% (wt/vol) BSA and 0.05% (vol/vol) Tween 20. Aliquots (20 µl) of the in vitro transcription-translation reaction mixture were used for each incubation. After extensive washing with TBS containing 0.05% Tween 20, the membranes were dried and exposed to a PhosphorImager (Molecular Dynamics).
Co-IP. Portions (200 µg) of nuclear extract from BJAB cells, as well as untreated or HU-treated HeLa cells, were incubated with either 8 µl of anti-hp150 antibody (MAb1) (42) or 2 µg of control immunoglobulin G (IgG) in immunoprecipitation (IP) buffer (20 mM HEPES [pH 7.5], 120 mM KCl, 5 mM MgCl2, 0.1% [vol/vol] NP-40, protease inhibitors) at 4°C for 3 h in a total reaction volume of 200 µl. Then 40 µl of IP buffer-equilibrated protein G-Sepharose beads (Amersham Pharmacia Biotech) was added, and the mixture was incubated for 3 h at 4°C. The beads were washed five times with wash buffer (IP buffer supplemented with an additional 20 mM KCl) before the protein complexes bound to the beads were eluted and split into two portions for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 10-µg portion of nuclear extract was used as the input control. Western blot analysis with an anti-BLM antibody, IHIC33 (53), and an anti-hp150 polyclonal antibody (gifts from Geneviève Almouzni) were used to detect proteins immunoprecipitated by MAb1. Enhanced chemiluminescence was performed as specified by the manufacturer (Amersham Pharmacia Biotech). Two other anti-hp150 antibodies were also used in the IP assays: MAb anti-CAF-1 p150 (Novus Biologicals) and Ab-3 (Oncogene Research Products). C-18 (anti-BLM; Santa Cruz) was used for the reverse IP in the above-described IP buffer containing 100 to 150 mM KCl. Nuclear extracts from BS cells were used as a negative control for the C-18 reverse IP.
Cellular fractionation. Exponentially growing HeLa cells, BS cells (GM08505), and BS cells containing the BLM cDNA (PSNF5 cells) (53) were harvested in phosphate-buffered saline (PBS) and washed twice with buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 mM dithiothreitol [DTT], protease inhibitors) before the pellet was resuspended in 3 volumes of buffer A+ (buffer A containing an additional 0.1% NP-40). After incubation for 5 min, the cells were centrifuged and the pellet was resuspended in 3 volumes of buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 25% [vol/vol] glycerol, 1 mM PMSF, 5 mM DTT, other proteinase inhibitors) and incubated on ice for 15 min. Finally, after the mixture was pelleted, the supernatant was taken as the soluble fraction of nuclear proteins (the concentration of NaCl was adjusted to 120 mM with buffer D, which contains 20 mM HEPES [pH 7.9], 1 mM PMSF, 5 mM DTT, and proteinase inhibitors) and the pellet was either subjected to ultrasonication to dissolve the insoluble fraction of nuclear proteins in SDS sample buffer or digested with DNase I in DIG buffer (50 mM NaCl, 10 mM Tris [pH 6.8], 3 mM MgCl2, 1 mM EGTA, 0.5% Triton, 100 µg of DNase I per ml, 50 µg of RNase A per ml, 1.2 mM PMSF) for 30 min at room temperature before the chromatin fraction was separated from the nuclear matrix fraction.
Cell synchronization and immunofluorescence microscopy. HeLa cells, BS cells, and PSNF5 cells (BS cells containing the BLM cDNA [BLM–/– + pBLM]) were grown on chamber slides (Lab-Tek) to up to 50% confluence before being subjected to synchronization or treatment with variant DNA-damaging agents. For a G1/S block, 5 mM thymidine and 2 µg of aphidicolin per ml were added to the cells for 28 and 24 h, respectively; for S-phase synchronization, 2 mM HU was used for 24 h; for a G2/M block, 1 µg of nocodazole per ml was incubated with the cells for 24 h. Synchronized cells or cells taken 20 h after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 4 to 18 h after UV treatment were permeabilized with 0.5% (vol/vol) Triton in PBSS (PBS supplemented with 5 mM MgCl2) plus 5 mM PMSF for 5 min at room temperature before being fixed in 2% paraformaldehyde for 20 min. After 20 min of further treatment with 0.5% (vol/vol) Triton, and following washing with PBSST (PBSS with 0.1% [vol/vol] Tween 20), the slides were blocked for 20 min in PBSST plus 5% (wt/vol) BSA before being incubated with the primary antibodies anti-BLM (1:200) (32), anti-RPA (1:50; Ab-1 [Oncogene Research Products]), anti-BRCA1 (1:100; Ab-1 [Oncogene Research Products]), anti-hp150 polyclonal antibody (1:2,000), and anti-hp150 MAb1 (1:100; Ab-3 [Oncogene research products]) for 2 h at room temperature. To visualize the primary antibodies, Cy3-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch) and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)-coupled goat anti-mouse IgG (1:100; ANAWA) or fluorescein isothiocyanate-coupled goat anti-mouse IgG (1:100; Jackson ImmunoResearch) were used, and 1 µg of 4,6-diamidino-2-phenylindole (DAPI) per ml was added to stain the DNA. The stained cells were mounted in Vectashield (Vector Laboratories) and observed under a Leica TCS 4D confocal microscope. Imaris software was used to process the images.
Chromatin assembly assay.
An in vitro chromatin assembly assay with a human cell-free system was utilized to analyze the effect of recombinant BLM on DNA repair-coupled chromatin assembly (15). Briefly, UV (500 J/m2)-irradiated circular pBluescript plasmid DNA was incubated for 3 h at 37°C with cytosolic extract derived from HeLa cells (30) supplemented with purified recombinant CAF-1 complex or with nuclear extract from either HeLa or BS cells (47) in the presence or absence of recombinant native BLM or BLM preincubated with anti-BLM antibody (32). The extent of supercoiling was examined by agarose gel electrophoresis followed by drying of the gel and exposure to X-ray film. Incorporation of [-32P]dCTP indicates newly synthesized DNA after nucleotide excision repair.
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RESULTS |
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To gain insight into the BLM-hp150 interaction in vivo, we performed co-IP experiments with nuclear extracts of human BJAB cells, using a MAb directed against hp150 (Fig. 1D). Endogenous BLM was effectively coprecipitated by the hp150 antibody (Fig. 1D, lower panel, lane 3) but was not detected when the hp150 antibody was omitted (lane 2). Similar results were obtained with two other anti-hp150 antibodies (data not shown). The reciprocal co-IP experiment was also carried out, in which an anti-BLM polyclonal antibody (C-18; Santa Cruz) was used to immunoprecipitate hp150 from HeLa nuclear extracts. As shown in Fig. 1D, hp150 could be specifically coimmunoprecipitated with anti-BLM (middle panel, lanes 6 and 7) but not with the control IgG antibody (lane 5). The results of this co-IP experiment have indicated that in soluble HeLa nuclear cell extracts, less than 10% of total hp150 is associated with BLM (data not shown, available upon request). Moreover, in addition to hp150, anti-BLM antibody efficiently coprecipitated topoisomerase III (Fig. 1D, bottom panel, lane 6), a protein known to interact with BLM (53). Importantly, the anti-BLM antibody (C-18) could not immunoprecipitate hp150 from BS (BLM–/–) cells (Fig. 1E, lane 3) whereas it efficiently immunoprecipitated hp150 from BS cells containing the BLM cDNA (BLM–/– + pBLM) (lane 4). We conclude from these different protein interaction experiments that BLM associates with hp150 of CAF-1 in yeast, in vitro, and in human cells.
Mapping of interaction regions on BLM and hp150. To identify the region of BLM that mediates the interaction with hp150, we generated a series of BLM deletion mutants. These deletion mutants were tested for interaction with the full-length hp150 in the YTH assay (Fig. 2A). Two independent regions in BLM were found to interact with hp150. The first interacting region resides at the N terminus of BLM and comprises amino acids 131 to 333, and the second region is located in the middle of the helicase domain and comprises amino acids 782 to 952 of BLM (Fig. 2A). The relatively weaker interaction of BLM amino acids 131 to 333 and BLM amino acids 782 to 952 with hp150 compared to that of full-length BLM could be due to ineffective folding of these two short forms. A similar YTH approach combined with an in vitro binding assay was used to map the BLM interaction region on hp150 (Fig. 2B). One region of hp150, comprising amino acids 551 to 714 containing the ED domain (amino acids 564 to 641) (24) and the dimerization domain (amino acids 642 to 678) (39), was found to be necessary and sufficient for interaction with BLM. Interestingly, this region is different from the region in hp150 that interacts with proliferating-cell nuclear antigen in vitro (33) and from the region critical for the interaction with p60, another subunit of CAF-1 (24).
BLM and hp150 colocalize on DNA replication block or DNA-damaging signals. Next, we analyzed whether BLM and hp150 colocalize within the nucleus of intact human cells by indirect immunofluorescence of exponentially growing HeLa cells. Previous studies using HeLa cells have shown that CAF-1 is present at replication foci, since it localizes at sites of 5-bromodeoxyuridine incorporation (25, 45). In these cells, BLM forms nuclear foci corresponding to PML nuclear bodies (21, 52). The observation of nuclear foci containing BLM with PML, RPA, and 5-bromodeoxyuridine led to the suggestion that these sites represent sites of DNA synthesis and repair (6). In untreated HeLa cells, we detected some nuclear foci that were enriched in both BLM and hp150. Under these conditions, only 7% of the cells contained a mean of 1.8 colocalizing BLM and hp150 foci per nucleus (Fig. 3A and Table 1). This observation suggested that the two proteins might associate at sites of endogenous damage during the replication process. We therefore asked whether the number of colocalizing BLM-hp150 foci increased in response to treatment of the cells with HU. Indeed, following a 20-h HU treatment, the proportion of cells containing BLM-hp150 colocalizing nuclear foci increased significantly, such that 40% of the population contained a mean of 7.6 colocalizing foci per nucleus (Fig. 3A; Table 1). We then tested whether other DNA-damaging agents resulted in increased BLM-hp150 colocalization. At 4 h after treatment of HeLa cells with UV light (50 J/m2), 25% of the cells contained a mean of 4.2 BLM-hp150 colocalizing foci per nucleus. In addition, after exposure of HeLa cells to MNNG for 20 h, 36% of the HeLa cells contained a mean of 6.9 BLM-hp150 colocalizing foci (Fig. 3A; Table 1), demonstrating that this significant increase in BLM-hp150 colocalization is irrespective of the type of DNA damage present.
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BLM-hp150 colocalization is cell cycle dependent. Since the subcellular localization patterns for BLM and CAF-1 are not identical during a normal, unperturbed cell cycle (25, 55), we set out to determine the stage of the cell cycle at which BLM and hp150 colocalize to the greatest extent. For this purpose, we synchronized HeLa cells in the G1/S, early S, and G2/M phases (Fig. 4A) and immunostained the cells with anti-BLM and anti-hp150 antibodies. Figure 4B and C show the pattern of BLM-hp150 colocalization in G1/S- and S-phase cells, respectively. As before, only a portion of cells exhibited BLM-hp150 colocalizing foci: only 3% of the G1/S phase cells contained BLM-hp150 colocalizing foci, while 38% of the S-phase cells displayed BLM-hp150 colocalization. During the G2/M stage, no cells showed any BLM-hp150 colocalization (data not shown). This reveals that colocalization between BLM and hp150 occurs most frequently during the S phase, where the majority of DNA replication and repair events are occurring.
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We also tested how p150 affects BLM helicase activity. Although hp150 inhibited BLM-catalyzed DNA unwinding, this appeared to be a nonspecific effect, probably mediated via the binding of hp150 to the DNA substrate, because hp150 also inhibited UvrD (data not shown, available on request).
BLM is required for an optimal CAF-1 response to HU. To further study the in vivo functional relationship between BLM and CAF-1, we examined CAF-1 function in BLM-deficient cells. To determine whether CAF-1-mediated chromatin assembly is affected in BS cells, we prepared nuclear extracts from BS cells and compared them with the nuclear extracts from wild-type (HeLa) cells for their ability to mediate chromatin assembly. The presence and absence of BLM in these cell lines was controlled by Western blotting (Fig. 6C). As shown in Fig. 6A, the endogenous CAF-1 activity was not obviously altered in BS cells (lane 2) compared with the CAF-1 activity from wild-type cells (lane 1).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results show that the proportion of detergent-resistant BLM and CAF-1 foci that colocalize in the nucleus increases after replication fork inhibition and treatment of cells with DNA-damaging agents. Together with the nuclear fractionation experiments, this suggests that the function of the BLM-hp150 interaction is associated with the matrix fraction of nuclear proteins. The increase in the number of foci containing both BLM and hp150 after HU treatment suggests that the BLM-hp150 complex concentrates in the vicinity of stalled replication forks. These replication forks probably contain nonduplex DNA structures, which might provoke a DNA damage response. In addition, BLM and hp150 are recruited together to discrete foci after DNA damage induction with MNNG or UV light. One interpretation of these phenomena is that in each case, the foci represent DNA repair processes that are initiated at a replication fork. In the case of HU treatment, as well as of UV- and MNNG-induced damage, stalled DNA replication may also be accompanied by a recombinational DNA damage response. The colocalization of BLM and hp150 at sites containing RPA and BRCA1 further supports the contention that the BLM-hp150 complex plays a repair role during DNA synthesis. Although the results of our indirect immunofluorescence experiments show that BLM is not required for recruitment of CAF-1 to nuclear foci, BLM seems to be required for an optimal CAF-1 response to HU. That the physical interaction between BLM and hp150 is of functional significance is indicated by the inhibitory effect of BLM on CAF-1-mediated chromatin assembly coupled to repair. This inhibitory effect appeared specific because a control helicase, UvrD, was unable to inhibit chromatin assembly.
Given all these observations, we propose that the BLM-hp150 interaction is important during times when DNA synthesis is perturbed in human cells. On the basis of our results, we propose a model suggesting a functional link between BLM and hp150 in restoring functional replication forks following fork stalling. Progression of a replication fork might be impeded by DNA secondary structures such as G-quadruplexes and DNA hairpins, thus causing the regression of the fork and formation of a four-way junction, a structural analog of a Holliday junction. It has been suggested previously that BLM and other RecQ helicases process such lesions that arise at replication forks (or are generated by the translocating fork itself) and, in doing so, reset the fork by "reverse" branch-migrating the four-way junction. This has the effect of preventing cleavage of the four-way junction and hence suppressing subsequent "promiscuous" recombination (10, 19, 23, 51). A second potential role for BLM at replication forks has been suggested by our recent observation that BLM and topoisomerase III together catalyze the dissolution of a recombination intermediate containing a double Holliday junction (54). Such double Holliday junctions are predicted to arise during postreplication gap filling when lesions on the lagging-strand template are bypassed. It is known that CAF-1-dependent chromatin assembly occurs on newly replicated DNA (43) and that CAF-1 is required in vitro for chromatin assembly following DNA repair (15). CAF-1 binds directly to PCNA, a DNA polymerase processivity factor that forms a sliding clamp around DNA, and the CAF-1-PCNA interaction promotes chromatin assembly (33). Although it remains unclear exactly how BLM and CAF-1 act after damage at a replication fork, our data suggest that the recruitment of BLM, probably not by CAF-1, may inhibit CAF-1, and hence chromatin assembly, in the vicinity of a stalled replication fork, perhaps to allow more efficient lesion processing. We envisage that such inhibition would continue until the point where normal replication structures are restored. Once the DNA lesion is bypassed and the replication fork is restored, the CAF-1 complex can be reactivated (it again assembles the newly synthesized DNA into chromatin) and DNA replication fork progression can resume.
This sequential scheme requires a level of regulation that may be achieved by posttranslational modification. Interestingly, BLM is phosphorylated following DNA damage (1, 2, 13), and UV damage induces changes in the amount of chromatin-bound phosphorylated CAF-1 complex (30). It is possible that either of these phosphorylation events or some other posttranslational modification(s) could be responsible for modulating both physical and functional interactions between BLM and CAF-1. The inhibitory effect of BLM on CAF-1 might also be modulated by associations with other interacting proteins. A candidate would be PCNA, which can bind to both CAF-1 (33) and BLM (S. L. Davies, J.-L. Li, and I. D. Hickson, unpublished data).
While the precise biochemical relationship between BLM and CAF-1 remains to be fully elucidated, the physical and functional interactions documented here provide a basis to further understand how BLM and hp150 function together to facilitate DNA repair and/or replication.
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ACKNOWLEDGMENTS |
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The IS group is financed by grants from the Stiftung für medizinische Forschung, Novartis Foundation, Olga Mayenfish Foundation, Sassella Foundation, Gebert Rüf Foundation, EU grant HPRN-CT-2002-00240, the Swiss Cancer League (OCS-01310-02-2003), the Swiss National Science Foundation (grants 31-58798.99 and 3100A0-100256/1), and the Zürcher Krebsliga. C.Z.B., J.-L.L. and I.D.H. are supported by Cancer Research UK and EU grant HPRN-CT-2002-00240. C.Z.B. was a Marie Curie Fellow of the European Union (HPMF-CT-2000-00952).
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FOOTNOTES |
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10 µm. (B) The amount of BLM and hp150 increases after HU treatment in the insoluble fraction of nuclear proteins. Untreated (lanes 1 and 3)and HU-treated (lanes 2 and 4) HeLa nuclear proteins were resolved into soluble (lanes 1 and 2) and insoluble (lanes 3 and 4) fractions, and these were subjected to SDS-PAGE followed by Western blotting for BLM (top panel), hp150 (middle panel), and SP1 (bottom panel [loading control]). (C) The increased amount of BLM and hp150 is mainly associated with the nuclear matrix. After DNase 1 digestion, the insoluble nuclear fraction was separated into two fractions: chromatin (lanes 1 and 2) and nuclear matrix (lanes 3 and 4). Lanes 1 and 3 contain untreated HeLa cells; lanes 2 and 4 contain HU-treated HeLa cells. Lamin B is shown as a fractionation and loading control. (D) BLM and hp150 colocalize with BRCA1 after HU treatment of HeLa cells. Red foci represent either BLM or hp150, and green foci reveal BRCA1 staining. DNA is revealed by DAPI staining. Bars, 
