Accumulation of Checkpoint Protein 53BP1 at DNA Breaks Involves Its Binding to Phosphorylated Histone H2AX*

Irene M. Ward {ddagger} §, Kay Minn {ddagger}, Katherine G. Jorda ¶ and Junjie Chen {ddagger} ||

From the {ddagger}Department of Oncology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 and the Division of Oncology, Columbia University, New York, New York 10032

Received for publication, March 14, 2003 , and in revised form, April 15, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The DNA of eukaryotic cells is constantly exposed to endogenous and exogenous DNA-damaging agents. To prevent the accumulation of genomic damage and avert cellular dysfunction, cells have evolved complex response mechanisms. 53BP1 was initially identified as a protein that binds to the central DNA binding domain of p53 and enhances p53-mediated transcriptional activation (1, 2). In response to genotoxic stress, 53BP1 rapidly redistributes from a diffuse nuclear localization into distinct nuclear foci suggesting that 53BP1 is involved in the DNA damage response (3, 4, 5, 6). Moreover the C terminus of 53BP1 contains two BRCT domains, a motif found in a number of proteins implicated in various aspects of cell cycle control, recombination, and DNA repair (7, 8). Subsequent studies have shown that 53BP1 becomes hyperphosphorylated in response to ionizing radiation (IR)1 and colocalizes with phosphorylated histone H2AX ({gamma}-H2AX) at the sites of DNA lesions (3, 4). Other proteins known to be involved in the DNA damage signaling pathway (i.e. BRCA1, Rad51, NBS1, and TopoBP1) were also found to colocalize with 53BP1 in these inducible foci (3, 4, 6, 9). Direct evidence for an important role of 53BP1 in the DNA damage response came recently from studies using 53BP1-deficient cells. Human cell lines treated with specific small interfering RNA to silence 53BP1 expression exhibited a defect in the intra-S phase checkpoint and, at low IR doses, a partial defect in the G2-M checkpoint (10, 11, 12). Moreover 53BP1-deficient mice are hypersensitive to ionizing radiation and show an increased incidence of developing thymic lymphomas (13). Several lines of evidence suggest that 53BP1 is a substrate of ATM, the kinase mutated in the human disease ataxia-telangiectasia, and is involved in the phosphorylation of various ATM substrates (3, 6, 10).

Despite this recent progress made toward 53BP1 function little is known about the initial activation of 53BP1. Recruitment of 53BP1 to {gamma}-H2AX foci seems to be a crucial step. H2AX-deficient cells lack normal 53BP1 foci formation and, like 53BP1-deficent cells, manifest a G2-M checkpoint defect after exposure to low doses of ionizing radiation (12). Moreover, H2AX–/– mice show a radiation sensitivity similar to 53BP1–/– mice (14). In this study we mapped the region required for 53BP1 foci formation in response to DNA damage. We show that a region upstream of the BRCT motifs is sufficient for 53BP1 foci formation and that this region interacts directly with phosphorylated H2AX. Using H2AX-deficient cells retransfected with either wild-type H2AX or an H2AX phosphomutant we confirm that phosphorylation of H2AX at Ser-140 is required for 53BP1 accumulation at DNA break areas. In contrast, radiation-induced phosphorylation of 53BP1 by ATM is not essential for the recruitment of 53BP1 to foci and occurs independently.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs and Transfection—53BP1 deletion mutants were generated by inserting stop codons and/or restriction sites at various positions into pCMH6K 53BP1 (2) using the QuikChange site-directed mutagenesis kit (Stratagene). A 3xNLS (nuclear localization sequence) was inserted into the NheI site upstream of the N-terminal hemagglutinin (HA) and His6 tags. U2OS cells were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. H2AX was PCR-amplified from human genomic DNA and inserted between the HA tag and an internal ribosomal entry site fused to the puromycin gene of a modified pcDNA3 vector. The H2AX phosphomutant was generated by replacing Ser-140 of H2AX with an alanine using the QuikChange mutagenesis kit (Stratagene). H2AX-deficient embryonic stem (ES) cells (provided by C. Bassing, Ref. 15) were transfected by electroporation.

Antibodies—Anti-S6P, anti-S25P/29P, and anti-S784P specific antibodies were generated by coupling synthetic 53BP1 peptides (S6P, CDPTG(P)SQLD; S25P/29P, CIED(P)SQPE(P)SQVLEDD; S784P, CSD(P)SQSWEDI where (P)S represents phosphoserine) to KLH using Imject maleimide-activated mcKLH (Pierce) prior to immunizing rabbits (Cocalico Biological). The antibodies were affinity-purified on agarose columns coupled with the non-phosphorylated or phosphorylated peptide (SulfoLink Coupling Gel, Pierce). Anti-53BP1 and anti-{gamma}-H2AX antibodies were generated as described previously (3). Monoclonal antibody HA11 specific for HA was purchased from BabCO Berkeley Antibody Co. Anti-ATM antibody Ab3 was purchased from Oncogene Research Products.

Immunofluorescence Staining, Immunoblots, and Immunoprecipitation—Cells grown on coverslips were fixed with 3% paraformaldehyde 1 h after exposure to 0 or 1 Gy of IR. After permeabilization with 0.5% Triton X-100, cells were blocked with 5% goat serum and incubated successively with the primary and secondary antibodies, each for 25 min at 37 °C. In case of DNase or RNase treatment, cells were irradiated, permeabilized with 0.5% Triton X-100 for 3 min, and incubated with either DNase I (10 units/ml) or RNase A (50 µg/ml) in phosphate-buffered saline plus calcium and magnesium for 30 min at 37 °C prior to fixation with 3% paraformaldehyde. Immunoprecipitation and immunoblot assays were done as described previously (3).

ATM Kinase Assays—ATM was precipitated from K562 cells, and aliquots of the ATM-protein A-Sepharose immunocomplex were resuspended in 25 µl of kinase buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 10 nM ATP). ATM kinase reactions were carried out at 30 °C for 20 min with 10 µCi of [{gamma}-32P]ATP and 1 µg of 53BP1 GST fusion proteins.

GST Pull-down Assays—GST pull-down experiments were performed by incubating 3 µg of various GST-labeled 53BP1 fragments with C-terminal H2AX peptide that was either phosphorylated or unphosphorylated at Ser-140 (CKATQA(P)SQEY) and had been immobilized on SulfoLink Coupling Gel (Pierce). Bound GST proteins were isolated by incubating the mixture for 1 h at 4 °C in 200 µl of NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8), 0.5% Nonidet P-40), washing five times with NETN, eluting the proteins with 2x Laemmli buffer, separating them by SDS-PAGE, and immunoblotting with horseradish peroxidase-conjugated anti-GST (B-14, Santa Cruz Biotechnology).

Generation of 53BP1-deficient Embryonic Cells—A 53BP1-deficient embryonic cell line was derived from 53BP1–/– blastocysts using a standard procedure. The generation of 53BP1-deficient mice is described in Ref. 13.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A Region Upstream of the Tandem BRCT Motif Is Required and Sufficient for 53BP1 Foci Formation—53BP1 is a large 1972-aa nuclear protein with a C-terminal tandem BRCT motif. Upstream of the BRCT repeats resides a bipartite nuclear localization signal (predictNLS, Ref. 16) and a tudor domain (RPS-BLAST, Ref. 17), a motif found in several RNA-binding proteins. In response to IR, 53BP1 rapidly redistributes to distinct nuclear foci that colocalize with {gamma}-H2AX (3, 4, 6). Treatment of irradiated cells with DNase, but not RNase, completely abolished 53BP1 and {gamma}-H2AX foci formation confirming that these foci localize to DNA (Fig. 1A).



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FIG. 1.
A region upstream of the BRCT domains is required and sufficient for damage-induced focus localization of 53BP1. A, DNase but not RNase treatment abolishes 53BP1 and {gamma}-H2AX foci formation in response to 1 Gy of IR. B, schematic diagram of the wild-type or mutant 53BP1 constructs that were N-terminally fused to a 3xNLS and an HA tag. Their abilities to form damage-induced foci is indicated by a +. KLR refers to the kinetochore localization region. C, U20S cells transiently expressing the HA-53BP1 constructs were irradiated with 1 Gy and immunostained 1 h later with anti-HA and anti-{gamma}-H2AX antibodies. PBS, phosphate-buffered saline.

 

To determine the minimal region required for the recruitment of 53BP1 to damage-induced foci, we generated various HA-tagged 53BP1 deletion mutants and examined their distribution in transiently transfected U2OS cells (Fig. 1, B and C, and data not shown). A 3xNLS motif fused to the N terminus of 53BP1 ensured nuclear expression of the various constructs. Truncation of the 53BP1 N terminus ({Delta}1–1052) or the BRCT domains ({Delta}1700–1972) did not affect 53BP1 foci formation as assessed by IF 1 h after exposure to 1 Gy of IR (Fig. 1C). However, increasing C-terminal deletions ({Delta}1305–1972 and {Delta}1052–1972) or deletion of a region upstream of the tandem BRCT motifs ({Delta}1052–1305) abolished 53BP1 foci formation (Fig. 2, B and C, and data not shown). Moreover a 53BP1 construct expressing residues 1052–1639 including the tudor domain was found to be sufficient for 53BP1 foci formation (Fig. 1, B and C) suggesting that the domain required for foci formation is contained within this region.



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FIG. 2.
53BP1 foci formation requires its binding to phosphorylated Ser-140 of H2AX. A, H2AX–/– ES cells were transiently transfected with either HA-tagged wild-type H2AX (HA-H2AXwt) or a S140A phosphorylation-deficient mutant (HA-S140A). 53BP1 foci formation was assessed by IF 1 h after exposure of cells to 1 Gy of IR. B, GST-53BP1 fragments, encoding different regions of 53BP1 as indicated, were incubated with immobilized C-terminal H2AX peptide that was either phosphorylated (P) or not at Ser-140. Pulled down proteins were separated by SDS-PAGE followed by immunoblotting with anti-GST antibodies. The addition of similar amounts of various GST proteins was verified by SDS-PAGE and Coomassie Blue staining. C, H2AX–/– cells were stained with anti-53BP1 antibodies and 4,6-diamidino-2-phenylindole, and mitotic cells were analyzed for kinetochore localization of 53BP1.

 

53BP1 Focus Localization Region Interacts Directly with {gamma}-H2AX—H2AX-deficient cells show greatly reduced 53BP1 foci formation implying that H2AX is involved in the recruitment of 53BP1 to radiation-induced foci (14). H2AX becomes phosphorylated at a conserved C-terminal SQ site upon exposure of cells to ionizing radiation (18). Phosphorylation of H2AX at Ser-140 is impaired in ATM-deficient cells suggesting that this site is dominantly phosphorylated by ATM (12). To analyze whether phosphorylation of H2AX at Ser-140 is required for 53BP1 redistribution we transiently expressed wild-type H2AX or a S140A phosphomutant in H2AX-deficient ES cells and assessed 53BP1 foci formation. As shown in Fig. 2A, expression of wild-type H2AX reconstituted 53BP1 foci formation in response to IR. In marked contrast, expression of the H2AX S140A phosphomutant was insufficient to induce 53BP1 accumulation at the sites of DNA strand breaks.

We had shown earlier that phosphorylated H2AX co-immunoprecipitates with 53BP1 upon exposure of cells to DNA damage (3). To determine whether the region required for 53BP1 focus localization interacts directly with {gamma}-H2AX, we used an in vitro pull-down assay. Six different 53BP1 GST fragments spanning the entire 53BP1 protein were incubated with immobilized C-terminal H2AX peptide that was either phosphorylated or non-phosphorylated at Ser-140. Only 53BP1 fragment 956–1354, which overlaps with the mapped focus localization region, showed strong interaction with the phosphorylated H2AX peptide (Fig. 2B). As a control, no binding was detected to the non-phosphorylated peptide bearing identical sequence (Fig. 2B).

Since H2AX directs 53BP1 accumulation in response to DNA damage, we asked whether H2AX is also required for the kinetochore localization of 53BP1 in mitotic cells (19). As shown in Fig. 2C, 53BP1 can be readily detected at the kinetochores in H2AX-deficient mitotic cells suggesting that the kinetochore localization of 53BP1 is not mediated by phospho-H2AX.

Phosphorylation of 53BP1 Is Not Required for Foci Formation—We had previously demonstrated that 53BP1 becomes hyperphosphorylated in response to IR, and three regions at the N terminus of 53BP1 were found to be phosphorylated by ATM in vitro (3). To map the phosphorylation sites we designed a series of GST fusion peptides containing one or two ATM binding motifs (SQ or TQ sites). ATM kinase assays using these purified GST fusion proteins as substrates, and ATM kinase immunoprecipitated from either K562 lysates (containing wild-type ATM) or ATM-deficient GM03189D lysates revealed peptides aa 1–12, aa 18–37, and aa 778–791 as putative ATM substrates (Fig. 3A). To examine whether the respective SQ sites become phosphorylated in vivo, we raised polyclonal antibodies against phosphorylated Ser-6 (anti-S6P), phosphorylated Ser-25 and Ser-29 (anti-S25P/29P), and phosphorylated Ser-784 (anti-S784P). All affinity-purified antisera recognized 53BP1 in irradiated cells but not in untreated controls when assessed by immunofluorescence analysis (data not shown). In addition, anti-53BP1 S25P/29P antibodies detected 53BP1 from irradiated ATM wild-type but not ATM-deficient cells by Western blot analyses (Fig. 3B). Pretreatment with {lambda}-phosphatase abolished the antibody binding further validating that anti-53BP1 S25P/29P specifically recognizes the phosphorylated form of 53BP1 (Fig. 3B).



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FIG. 3.
Damage-induced phosphorylation of 53BP1 occurs independently of 53BP1 foci formation. A, in vitro mapping of potential ATM phosphorylation sites. ATM kinase assays were performed using ATM kinase immunoprecipitated from K562 lysates (ATM wild type) or GM03189D lysates (ATM-deficient) and GST fusion peptides containing one or two SQ or TQ sites of 53BP1. GST or GST fusion protein containing 14 amino acids surrounding the Ser-15 of p53 (p53S15) were used as negative and positive controls, respectively. B, 53BP1 is phosphorylated in vivo following IR. 293T cells containing wild-type ATM and ATM-deficient GM03189D cells were mock-treated or irradiated (50 Gy). After 1 h, whole cell lysates were immunoprecipitated with anti-53BP1 antibody. Duplicate samples were treated with or without {lambda}-phosphatase ({lambda}PPase). Western blots were performed with anti-53BP1 or an anti-phospho-53BP1 antiserum raised against the phosphoserines 25/29 of 53BP1. C, ATM-dependent 53BP1 phosphorylation is not required for 53BP1 foci formation. HA-tagged full-length 53BP1 (FL) or a phosphorylation-deficient mutant of 53BP1 (4SA) were stably expressed in 53BP1-deficient embryonic cells. 53BP1 expression levels were assessed by immunoprecipitation. 53BP1 foci formation was analyzed by IF 1 h after 1 Gy of IR. D, 53BP1 foci formation is not required for ATM-dependent phosphorylation of 53BP1 following DNA damage. 53BP1-deficient embryonic cells were transiently transfected with HA-tagged full-length 53BP1 or a deletion mutant lacking part of the focus formation region. 53BP1 localization and ATM-dependent phosphorylation was analyzed 1 h after 1 Gy of IR by co-staining with anti-HA antibody and anti-phospho-53BP1 antiserum (anti-25P/29P). E, an aliquot of the transfected cells was either mock-treated or irradiated with 10 Gy. 53BP1 was immunoprecipitated from cell lysates, and phosphorylation of 53BP1 was analyzed by immunoblotting with the 53BP1 phosphospecific anti-S25P/29P antibodies. wt, wild type; IP, immunoprecipitation.

 

To test whether phosphorylation of 53BP1 is required for the recruitment of 53BP1 to sites of DNA lesions, we generated a phosphorylation-deficient mutant (53BP1 4SA) by mutating the mapped ATM target sites (Ser-6, Ser-25/Ser-29, and Ser-784) to alanines. 53BP1–/– embryonic cells transfected with this phosphomutant showed normal 53BP1 foci formation in response to IR (Fig. 3C), indicating that ATM-dependent phosphorylation of 53BP1 is not required for recruitment to or retention of 53BP1 at DNA break sites.

Phosphorylation of 53BP1 might occur at the break areas. To test this possibility, we transiently transfected 53BP1-deficient embryonic cells with the HA-tagged mutant that lacks part of the 53BP1 focus localization region ({Delta}1052–1305) and remains a diffuse nuclear localization upon exposure of cells to IR. Co-immunostaining with anti-HA and anti-53BP1 S25P/29P antibodies revealed that ATM-dependent 53BP1 phosphorylation does not require 53BP1 foci formation (Fig. 3D). Immunoprecipitation assays confirmed that 53BP1 {Delta}1052–1305 becomes readily phosphorylated at Ser-25/Ser-29 in response to IR (Fig. 3E). These findings are consistent with a recent report describing phosphorylation of 53BP1 Ser-25 in H2AX-deficient cells (12). Taken together, these results suggest that 53BP1 focus localization and ATM-dependent phosphorylation of 53BP1 are regulated independently.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon exposure of cells to genotoxic stress, 53BP1 rapidly redistributes from a pan-nuclear localization to distinct nuclear foci at the sites of DNA strand breaks. In this study we have mapped the region required for 53BP1 foci formation and examined the role of H2AX in 53BP1 accumulation. Moreover, we provided evidence that phosphorylation of 53BP1 by the ATM kinase occurs independently of 53BP1 foci formation.

53BP1 had been speculated to be involved in the DNA damage response based on its C-terminal tandem BRCT domains. This motif was first detected in the BRCA1 C terminus and has been reported to bind directly to DNA breaks (20). Surprisingly the BRCT domains of 53BP1 were found to be dispensable for 53BP1 foci formation. Instead a region upstream of the BRCT motifs proved to be essential for 53BP1 accumulation at sites of DNA strand breaks. Our data suggest that the damage-induced phosphorylation of H2AX directs 53BP1 accumulation at sites of DNA strand breaks. First, H2AX-deficient cells reconstituted with a H2AX phosphomutant failed to induce or sustain 53BP1 foci formation. Second, a 53BP1 fragment (residues 956–1354) contained within the 53BP1 focus localization region interacted strongly with phosphorylated H2AX in vitro. Third, 53BP1 co-immunoprecipitates with H2AX in a DNA damage-dependent manner (3). Thus, it is likely that the DNA damage-induced phosphorylation of H2AX at Ser-140 increases the interaction between H2AX and 53BP1 and leads to the accumulation of 53BP1 at the sites of DNA breaks.

Interestingly the focus localization region we mapped includes a region required for 53BP1 kinetochore localization in mitotic cells (residues 1220–1601) (19). Very recently, Morales and colleagues (21) showed that the kinetochore localization region is also essential for 53BP1 foci formation in response to DNA damage suggesting that both events might be regulated in a similar fashion. However, the kinetochore localization of 53BP1 is unlikely to involve DNA lesions (19). We have shown that 53BP1 kinetochore localization appears normal in H2AX-deficient cells, suggesting that kinetochore localization of 53BP1 is not mediated by phospho-H2AX. Further fine mapping studies will be necessary to clarify whether the same 53BP1 region initiates the recruitment of 53BP1 to DNA strand breaks or the kinetochore, respectively.

Phosphorylation by the ATM kinase plays a key role in the activation of various proteins involved in the DNA damage response (for example, see Ref. 22). A recent study by Bakkenist and Kastan (23) revealed that ATM forms an inactive oligomer in unirradiated cells. Upon radiation, ATM is rapidly autophosphorylated and dissociates from the complex thereby providing other substrates access to its kinase domain. Interestingly autophoshorylation and activation of ATM seem to occur at some distance to DNA break sites, and ATM then migrates in the nucleus to phosphorylate various substrates either at the break sites or elsewhere in the nucleus (23). This model is consistent with our finding that ATM-dependent phosphorylation of 53BP1 is not restricted to sites of DNA strand breaks and can occur within the entire nucleus. However, phosphorylation of 53BP1 alone is unlikely to trigger 53BP1 activation since deletion of the ATM target sites does not affect 53BP1 relocalization. Moreover the relocalization of 53BP1 appears to be required for efficient phosphorylation of ATM substrates at the sites of DNA breaks (data not shown). We therefore speculate that the rapid recruitment of 53BP1 to DNA break sites and the retention of 53BP1 at the sites of DNA breaks by binding to phospho-H2AX is one of the key steps in the activation of 53BP1 following DNA damage.


    FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health, Breast Cancer Research Foundation, and Prospect Creek Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a postdoctoral fellowship from the Department of Defense breast cancer research program. Back

|| Recipient of a Department of Defense breast cancer career development award. To whom correspondence should be addressed. Tel.: 507-538-1545; Fax: 507-284-3906; E-mail: Chen.junjie{at}mayo.edu.

1 The abbreviations used are: IR, ionizing radiation; {gamma}-H2AX, phosphorylated histone H2AX; ATM, ataxia-telangiectasia mutated; NLS, nuclear localization sequence; HA, hemagglutinin; ES, embryonic stem; KLH, keyhole limpet hemocyanin; Gy, gray(s); GST, glutathione S-transferase; aa, amino acid(s); IF, immunofluorescence; BRCT, BRCA1 C terminus. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Craig Bassing for the H2AXFlox/Flox and H2AX{Delta}/{Delta} ES cells. We also thank Drs. Larry Karnitz and Scott Kaufmann and members of the Chen and Karnitz laboratories for helpful discussions. We are grateful to the Mayo Protein Core facility for the synthesis of peptides and to the Mayo Gene Targeted Mouse Core facility for help with the generation of 53BP1-deficient mice and 53BP1-deficient ES cells.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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