Tan Lin

Dr Likui Zhang
email: lkzhang@yzu.edu.cn

Donghao Jiang

Leilei Wu

Kaige Chen

Li Li

Cuili Jin

Zheng Li

Dr Philippe Oger
email: philippe.oger@insa-lyon.fr

Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5

Keywords: Hyperthermophilic Archaea, Flap endonuclease 1, DNA replication and repair, Biochemical characteristics, Mutational studies

Flap endonuclease 1 (FEN1) plays important roles in DNA replication, repair, and recombination. Herein, we report the biochemical characteristics and catalytic mechanism of a novel FEN1 from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-FEN1). As expected, the recombinant Tb-FEN1 can cleave 5′-flap DNA. However, the enzyme has no activity on cleaving pseudo Y DNA, which sharply contrasts with other archaeal and eukaryotic FEN1 homologs. Tb-FEN1 retains 24% relative activity after heating at 100 o C for 20 min, demonstrating that it is the most thermostable among all reported FEN1 proteins. The enzyme displays maximal activity in a wide range of pH from 7.0 to 9.5. The Tb-FEN1 activity is dependent on a divalent metal ion, among which Mg 2+ and Mn 2+ are optimal. Enzyme activity is inhibited by NaCl. Kinetic analyses estimated that the activation energy for the removal of 5′-flap from DNA by Tb-FEN1 was 35.7 ± 4.3 kcal/mol, which is the first report on the energy barrier for excising 5′-flap from DNA by a FEN1 enzyme. Mutational studies demonstrate that the K87A, R94A and E154A amino-acid substitutions abolish cleavage activity and reduce 5′-flap DNA binding efficiencies, suggesting that residues K87, R94, and E154 in Tb-FEN1 are essential for catalysis and DNA binding as well.

Overall, Tb-FEN1 is an extremely thermostable endonuclease with unusual features.

Introduction

Flap endonuclease 1 (FEN1), a structure specific nuclease, catalyzes the removal of 5′-flap from 5′-bifurcated DNA structures during DNA replication, repair, and recombination [START_REF] Balakrishnan | Flap endonuclease[END_REF], making this endonuclease fundamental in several essential biological processes. Indeed, FEN1 has been shown to excise RNA primers to process the Okazaki fragments during DNA replication [START_REF] Bambara | Enzymes and reactions at the eukaryotic DNA replication fork[END_REF]. In the long-patch base excision repair, FEN1 plays a role in removing damaged nucleotides [START_REF] Klungland | Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1)[END_REF]. Furthermore, FEN1 can facilitate homologous recombination by removing divergent sequences at DNA break ends [START_REF] Kikuchi | FEN-1 facilitates homologous recombination by removing divergent sequences at DNA break ends[END_REF]. In addition, FEN1 has also been implicated in other pathways such as non-homologous end joining, stalled replication fork rescue, apoptotic DNA fragmentation, and α-segment error editing [START_REF] Wu | A role for FEN-1 in nonhomologous DNA end joining: the order of strand annealing and nucleolytic processing events[END_REF][START_REF] Zheng | Functional regulation of FEN1 nuclease and its link to cancer[END_REF][START_REF] Liu | Okazaki fragment maturation involves alpha-segment error editing by the mammalian FEN1/MutSalpha functional complex[END_REF]. Besides, FEN1 is also crucial for genomic stability in eukaryotes [START_REF] Dehé | Control of structure-specific endonucleases to maintain genome stability[END_REF][START_REF] Tsutakawa | Phosphate steering by flap endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability[END_REF]. Thus, FEN1 is a central component of DNA metabolism since it can be involved in various DNA metabolic pathways (Liu et al., 2004). Additionally, FEN1 can be used as a novel diagnostic, prognostic biomarker and therapeutic target for cancer and gene editing [START_REF] Varshney | DNA-guided genome editing using structureguided endonucleases[END_REF][START_REF] Xu | An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease[END_REF][START_REF] Guo | FEN1 endonuclease as a therapeutic target for human cancers with defects in homologous recombination[END_REF][START_REF] Zhao | Flap endonuclease 1 (FEN1) as a novel diagnostic and prognostic biomarker for gastric cancer[END_REF].

FEN1 is a member of the newly emerging family of structure-specific endonucleases that are evolutionarily ubiquitous in Archaea and Eukarya [START_REF] Lieber | The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination, and repair[END_REF][START_REF] Shen | Flap endonuclease homologs in archaebacteria exist as independent proteins[END_REF]. FEN1 possesses flap endonuclease activity as well as 5′→3′ exonuclease activity that allows it to remove RNA primers during lagging strand synthesis in DNA replication and damaged DNA fragments in various DNA repair pathways. Structural and biochemical data have provided a wealth of information about structure-function relationship of FEN1 [START_REF] Finger | The wonders of flap endonucleases: structure, function, mechanism, and regulation[END_REF][START_REF] Grasby | Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases[END_REF]. The crystal structures of FEN1 homologs demonstrate that a conserved helical arch harbors the active site [START_REF] Ceska | A helical arch allowing singlestranded DNA to thread through T5 5'-exonuclease[END_REF]Hosfield et al., 1998b;[START_REF] Hwang | The crystal structure of flap endonuclease-1 from Methanococcus jannaschii[END_REF][START_REF] Chapados | Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair[END_REF]. Besides, a flexible loop is essential for flap cleavage since the 5′-end of the DNA flap could thread through the hole of the loop tracking the length of the 5′-tail [START_REF] Murante | Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage[END_REF][START_REF] Storici | The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair[END_REF][START_REF] Chapados | Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair[END_REF][START_REF] Shi | Interplay of catalysis, fidelity, threading, and processivity in the exo-and endonucleolytic reactions of human exonuclease I[END_REF].

As one of the ancestors of Eukarya, Archaea are an ideally simplified model for many aspects of DNA replication and repair in Eukarya. Since the first FEN1 from the hyperthermophilic euryarchaeon Methanococcus jannaschii was structurally characterized (Hosfield et al., 1998a;[START_REF] Hwang | The crystal structure of flap endonuclease-1 from Methanococcus jannaschii[END_REF][START_REF] Rao | Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements[END_REF][START_REF] Bae | Expression, purification, characterization, and crystallization of flap endonuclease-1 from Methanococcus jannaschii[END_REF], several FEN1 homologs have been reported from other hyperthermophilic Archaea (HA), including Archaeoglobus fulgidus [START_REF] Chapados | Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair[END_REF]Hosfield et al., 1998a), Pyrococcus horikoshii [START_REF] Matsui | Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically[END_REF][START_REF] Matsui | Serial intermediates with a 1 nt 3'flap and 5' variable-length flaps are formed by cooperative functioning of Pyrococcus horikoshii FEN-1 with either B or D DNA polymerases[END_REF][START_REF] Matsui | Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii[END_REF], Pyrococcus furiosus (Hosfield et al., 1998a;Hosfield et al., 1998b;[START_REF] Allawi | Modeling of flap endonuclease interactions with DNA substrate[END_REF], Thermococcus kodakarensis [START_REF] Burkhart | The GAN exonuclease or the flap endonuclease Fen1 and RNase HII are necessary for viability of Thermococcus kodakarensis[END_REF][START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF], Desulfurococcus amylolyticus [START_REF] Mase | Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus[END_REF], Aeropyrum pernix [START_REF] Collins | A preliminary solubility screen used to improve crystallization trials: crystallization and preliminary X-ray structure determination of Aeropyrum pernix flap endonuclease-1[END_REF], Sulfolobus tokodaii [START_REF] Horie | The N-terminal region is important for the nuclease activity and thermostability of the flap endonuclease-1 from Sulfolobus tokodaii[END_REF] or Sulfolobus solfataricus [START_REF] Beattie | Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation[END_REF]. The FEN1 homologs from HA have two common characteristics: thermostability and thermophilicity, capable of cleaving DNA at high temperature. Like eukaryotic FEN1, the homologs from HA also utilize a two divalent metal ion mechanism for catalysis. These two divalent metal ions are bound by highly conserved acidic amino acids at the bottom of a positively charged cleft to form a complex, among which one metal ion is presumably responsible for catalysis and the other for substrate binding [START_REF] Shen | Essential amino acids for substrate binding and catalysis of human flap endonuclease 1[END_REF]. However, the FEN1 proteins from HA harbor characteristics distinct from the eukaryotic and other archaeal homologs, which comprise varied substrate specificity, tolerance to acidic pH and elevated temperatures, dependency to different metal ions and salt tolerance. For example, the M. jannaschii FEN1 functions effectively at high salt concentrations, which contrasts with the eukaryotic FEN1 [START_REF] Horie | The N-terminal region is important for the nuclease activity and thermostability of the flap endonuclease-1 from Sulfolobus tokodaii[END_REF]. Although the overall structures of FEN1s from several HA are similar to those of eukaryotic FEN1 proteins [START_REF] Matsui | Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii[END_REF][START_REF] Mase | Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus[END_REF], they possess subtle conformational differences. For instance, the conformation of the putative substrate-binding pocket of the FEN1 from the hyperthermophilic archaeon D. amylolyticus is unique and created by a helical clamp and a flexible loop [START_REF] Mase | Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus[END_REF]. The crystal structure of FEN1 from the hyperthermophilic archaeon P. horikoshii also demonstrates that its active cleft is formed by one large loop and four small loops [START_REF] Matsui | Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii[END_REF].

Relatively few FEN1 proteins from the hyperthermophilic genus Thermococcus have been investigated to date. Recent genetic studies have shown that the ∆fen1 T. kodakarensis mutant grows well, suggesting that FEN1 has no discernible effect on viability and growth of this archaeon [START_REF] Burkhart | The GAN exonuclease or the flap endonuclease Fen1 and RNase HII are necessary for viability of Thermococcus kodakarensis[END_REF]. Further biochemical data demonstrate that the T. kodakarensis FEN1 displays maximal activity for excising 5′flap from DNA at 80°C and at pH 7.5 [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF].

Thermococcus barophilus Ch5 is an anaerobic thermo-piezophilic archaeon, which was isolated from a deep-sea hydrothermal field of the Mid-Atlantic Ridge (Logachev field chimney, 3,020 m depth), growing optimally at 88 o C under 40 MPa of hydrostatic pressure (Kim et al. Nature 2010). T. barophilus Ch5 possesses a putative FEN1 [START_REF] Oger | Complete genome sequence of the hyperthermophilic and piezophilic archaeon Thermococcus barophilus Ch5, capable of growth at the expense of hydrogenogenesis from carbon monoxide and formate[END_REF]. In this work, we report the gene cloning, characterization, and mutational studies of a FEN1 from T. barophilus Ch5 (Tb-FEN1), showing that the enzyme is a quite thermostable endonuclease with unusual features. Importantly, we report for the first time the activation energy required for 5′-flap DNA removal by a FEN1 enzyme.

Materials and methods

Materials

Plasmid Extraction Kit, PCR Cycle Pure Kit, Gel Extraction Kit, and the competent Escherichia coli DMT cells were purchased from Transgene (Beijing, China); T4 DNA ligase, BamHI, and XhoI were purchased from Thermo Scientific (Waltham, MA). High-fidelity PCR Mix was purchased from Vazyme (Nanjing, China). Chemicals were purchased from Amresco (WA, USA).

Cloning, expression, and purification of Tb-FEN1

The Tb-FEN1 gene (TBCH5v1_1306) was amplified from T. barophilus Ch5 genomic DNA with High-Fidelity PCR Mix with the specific primers containing BamHI and XhoI restriction sites (Tb-FEN1 F and Tb-FEN1 R, Table 1). After DNA denaturation at 95°C for 3 min, the amplified PCR reaction was performed by following 34 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 5 min. After digestion with BamHI and XhoI, the amplified DNA product was ligated into the pET-28at-plus vector digested with the same two enzymes to produce a recombinant Tb-FEN1 gene with a 6 x His-tag at the C-terminus. The construction was verified by sequencing and then transformed into the expression strain E. coli BL21 (DE3) RIL cells for protein expression.

The Tb-FEN1 gene was expressed in LB medium with 100 μg/mL kanamycin and 34 μg/mL chloramphenicol by the addition of isopropyl thiogalactoside (IPTG) at a final concentration of 0.1 mM when the OD600 of the expression strain E. coli reached 0.6 at 37°C. The culture was further incubated for about 10 hrs at room temperature under agitation.

The culture was harvested by centrifugation (6000×g for 15 min at 25 o C) and the pellet was resuspended in Ni column buffer A (20 mM Tris-HCl pH 8.0, 1 mM dithiothreitol (DTT), 500 mM NaCl, 50 mM imidazole, and 10% glycerol). The cells were disrupted by sonication on ice and the supernatant was harvested into a 50 mL tube by centrifugation (16000×g for 30 min at 4°C). To remove non-thermostable E. coli nuclease activities, the supernatant was heated at 70°C for 20 min and then collected by centrifugation at 16000×g for 30 min at 4°C. The resulting supernatant was loaded into a HisTrap FF column (GE Healthcare, Uppsala, Sweden) using NCG™ Chromatography System (Bio-Rad, Hercules, CA, USA) and then the Tb-FEN1 protein was eluted in a linear gradient of 50-500 mM imidazole. The collected fractions harboring the Tb-FEN1 protein were verified by electrophoresis in a 12% SDS-PAGE.

The purified Tb-FEN1 protein was dialyzed against a storage buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, and 50% glycerol) and stored at -80°C. Based on the molar extinction coefficient of the Tb-FEN1 protein (43,890 M -1 cm -1 ), the Tb-FEN1 protein concentration was determined by measuring the absorbance at 280 nm.

Construction, expression, and purification of the Tb-FEN1 mutants

The Tb-FEN1 N30M, K87A, R94A, E154A, Q172E, Y174M, R186A, N187H, L226F, D236A and N238C mutants were engineered using the plasmid harboring the wild-type Tb-FEN1 gene as template with a Site-directed Mutagenesis Kit according to the manufacturer's instructions. The sequences of the mutagenic primers are listed in Table 1. After verification of the contructs by sequencing, the Tb-FEN1 mutant proteins were overexpressed, purified, and quantified as described above for the wild-type protein.

DNA cleavage assays

The oligonucleotides were synthesized by Sangon Biotech Company, China.

Oligonucleotide sequences are listed in Table S1. The Cy3-labeled oligonucleotide was annealed with the appropriate complementary oligonucleotides in buffer (20 mM Tris-Cl pH 8.0 and 100 mM NaCl) to prepare 5′-flap, pseudo Y DNA or dsDNA (Table S2).

The annealing reactions were performed at 100 o C for 3 min, and cooled down slowly to room temperature at least 4 hrs.

The standard Tb-FEN1 DNA cleavage reactions were performed in reactions (10 μL) containing 20 mM Tris-HCl pH 8.0, 1 mM DTT, 8% glycerol, 100 nM Cy3-labeled 5′-flap DNA and 1000 nM Tb-FEN1 at 45°C for 30 min, unless stated otherwise. 10 μL of stop solution containing 98% formamide and 20 mM EDTA (Ethylene Diamine Tetraacetic Acid) was added to stop the reactions. After denaturation at 95°C for 5 min and rapid cooling on ice for 5 min, the cleaved product was separated by electrophoresis in a denaturing 15% polyacrylamide gel containing 8 M urea in 0.5 × TBE (Tris-Borate-EDTA). Quantitative analysis was performed with the ImageQuant software after imaging the gels with a molecular image analyzer (PharosFx System, BioRad). All DNA cleavage assays were replicated three times.

Biochemical characterization assays

To investigate the biochemical characteristics of the 5′-flap DNA we used in this work, including its thermostability, optimal pH, divalent metal ion requirement and salt tolerance, we performed DNA cleavage reactions catalyzed with Tb-FEN1 in various conditions. To determine the thermostability of the enzyme, the Tb-FEN1 protein was heated at 85 o C, 90 o C, 95 o C and 100 o C for 20 min, respectively. After heating, the Tb-FEN1 protein was used to perform the DNA cleavage reactions under standard conditions.

The optimal pH-dependence of the Tb-FEN1 activity was examined by performing the DNA cleavage reactions at various pHs (6.5, 7.0, 7.5, 8, 8.5, 9, 9.5 and 10). The corresponding pHs were prepared using five different buffers (all at 20 mM concentrations) as follows: sodium phosphate-NaOH (pH 6.5, 7.0 and pH 7.5), Tris-HCl (pH 8.0 and 8.5), and Glycine -NaOH (pH 9.0, pH 9.5 and pH 10.0). The reaction products were treated as described above.

We investigated the effects of divalent metal ions on the Tb-FEN1 activity by performing the DNA cleavage reactions in presence of 5 mM of either Mg 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Co 2+ , Ni 2+ , or Cu 2+ . The reaction products were treated as described above.

We determined the effect of salinity on Tb-FEN1 activity by performing the DNA cleavage reactions at various NaCl concentrations ranging from 50 to 1000 mM. The reaction products were treated as described above.

DNA-binding assays

DNA-binding assays were performed by incubating the wild-type and mutant Tb-FEN1 with 100 nM 5′-flapped DNA in buffer (10 μL) containing 20 mM Tris-HCl pH 8.0, 1 mM DTT, and 8% glycerol at 25°C for 10 min. The free DNA substrate and bound DNA product with the wild-type and mutant Tb-FEN1 were separated by electrophoresis in a 4% native polyacrylamide gel in 0.1 × TBE buffer. Quantitative analysis was performed with the ImageQuant software after the gels were scanned with a molecular image analyzer (PharosFx System, BioRad). DNA binding assays were repeated three times.

Kinetic analysis

Under the single-turnover conditions where Tb-FEN1 (1000 nM) is 10-fold higher than 5′-flap DNA (100 nM), the DNA cleavage assays were performed at varied temperatures for various times. The calculated concentrations of the remaining substrate after DNA cleavage by Tb-FEN1 were fitted using KaleidaGraph (Synergy Software) by a single exponential decay equation:

[Remaining substrate] = A exp(-kendot)
where A and kendo are the reaction amplitude and the cleavage rate, respectively.

The measured kendo values at varied temperatures were treated to be Ln kendo, according to an Arrhenius Equation:

kendo =Ar [exp(-Ea/RT)]
where Ar, Ea, R, and T represent a proportionality constant, the activation energy, the universal gas constant, and reaction temperature in Kelvin, respectively. An activation energy was yielded by plotting the treated kendo values (Ln kendo) at varied temperatures with the treated reciprocal of reaction temperature (1000/K) by a linear regression.

Results

The genome of T. barophilus Ch5 encodes a putative FEN1

The genome of T. barophilus Ch5 encodes a putative flap endonuclease 1 (gene TBCH5v1_1306, accession number WP_056933915.1), belonging to the structurespecific nuclease superfamily. The sequence alignment of Tb-FEN1 with other archaeal and eukaryotic FEN1 homologs demonstrates that Tb-FEN1 displays 85%, 85%, 85%, 58%, 55%, 53%, 51%, 36%, 37% and 38% amino acid identity to the FEN1 homologs from P. horikoshii, P. furiosus, T. kodakarensis, A. fulgidus, D. amylolyticus, S. solfataricus, M. jannaschii, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens, respectively. As shown in Fig. 1A, Tb-FEN1 possesses several highly conserved amino acids residues in the conserved motifs characteristic of the FEN1 homologs from other Archaea and eukaryotes, confirming that Tb-FEN1 resembles other FEN1 homologs, and should be capable of removing 5′-flap from DNA.

To biochemically characterize the enzyme, we cloned the Tb-FEN1 gene into the pET-28at-plus expression vector and further expressed its product in the E. coli BL21(DE3) RIL cells. The recombinant Tb-FEN1 protein was successfully overexpressed with a 6 x His-tag at its C-terminus as a ~ 39 kDa protein (Fig. 1B).

Using sonication, heat treatment (70°C for 20 min), and affinity chromatography with a Ni column, the recombinant Tb-FEN1 protein was purified to homogeneity (Fig. 1B).

Tb-FEN1 cleaves specifically 5′-flap DNA

We next employed the purified Tb-FEN1 to investigate whether this enzyme can cleave DNA by performing the DNA cleavage reactions using various DNA substrates.

As shown in Fig. 2A, the 5′-flap DNA was gradually cleaved with increasing Tb-FEN1 protein concentrations at 45 o C up to 91% cleavage percentage in the presence of 1000 nM Tb-FEN1, showing that the enzyme can effectively cleave 5′-flap DNA. In contrast, no cleaved product was observed using the pseudo Y DNA, no matter the Tb-FEN1 concentration used (Fig. 2B), which differs from other reported FEN1 homologs from Archaea and eukaryotes. Similarly, Tb-FEN1 had no activity towards normal ssDNA and dsDNA (Figs. 2C and2D). Thus, these observations suggest that Tb-FEN1 can efficiently cleave 5′-flap DNA but not pseudo Y DNA.

Biochemical characteristics of Tb-FEN1

Since Tb-FEN1 can specifically cleave 5′-flap DNA, we employed the 5′-flap DNA as substrate to examine its thermostability, optimal pH, divalent metal ion requirement and salt tolerance. As shown in Fig. 3A, we demonstrated that the heated Tb-FEN1 protein retained 87%, 68%, and 71% cleavage activity after a 20 min treatment at 80 o C, 85 o C, and 90 o C, respectively. Even after heating at 100 o C for 20 min, Tb-FEN1 still possessed about 24% cleavage activity. Thus, Tb-FEN1 is a thermostable endonuclease.

Moreover, we found that Tb-FEN1 can cleave 5′-flap DNA in a pH range from 6.5 to 9.5 with variable efficiencies (6.0-10.0) (Fig. 3B), demonstrating an optimal activity over a large pH range (7.0 to 9.0). In contrast, no activity was detected when the cleavage reactions of Tb-FEN1 were performed at pH 10 and pH 11. This observation is to take with caution since it might also result from the instability of the 5′-flap DNA under the high alkaline condition. Compared with Tb-FEN1, T. kodakarensis FEN1 has a relative narrow pH optimum range around 7.5 [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF], while that of M. jannaschii FEN was between 6 and 7, and pH 8 for the murine FEN-1 (Harrington and Liebern, 1994). Thus, the pH requirements of archaeal and eukaryotic FEN1 homologs may vary greatly depending on the source organism.

Next, we investigated whether the Tb-FEN1 activity is dependent on a divalent metal ion as observed for other FEM1 proteins. In the control reaction without a divalent metal ion, Tb-FEN1 had only residual activity (Fig. 3C,CK). This activity was further slightly reduced in the presence of 10 mM EDTA (Fig. 3C, EDTA), which confirms that the enzyme activity is dependent on a divalent metal ion. Tb-FEN1 exhibited its maximal activity in the presence of Mg 2+ , and to almost the same extent in the presence of Mn 2+ , which is similar to what is observed for the murine FEN1 [START_REF] Zheng | Distinct roles of two Mg 2+ binding sites in regulation of murine flap endonuclease-1 activities[END_REF]. Tb-FEN1 exhibited an extremely weak activity in the presence of Cu 2+ , Zn 2+ or Ca 2+ and an intermediate activity in the presence of Co 2+ and Ni 2+ . In contrast to Tb-FEN1, Mn 2+ greatly reduces the M. jannaschii FEN1 activity [START_REF] Bae | Expression, purification, characterization, and crystallization of flap endonuclease-1 from Methanococcus jannaschii[END_REF], and inhibits that of T. kodakarensis FEN1 [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF]. Furthermore, while the T. kodakarensis FEN1 activity is highest in the presence of Co 2+ [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF], Tb-FEN1 retains only 25% relative activity with Co 2+ , and while Cu 2+ enables some activity with Tb-FEN1, it inhibits that of the T. kodakarensis FEN1 enzyme [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF]. Reversely, Tb-FEN1 is inactive in the presence of Zn 2+ , whereas it can activate the T. kodakarensis FEN1 activity [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF].

Interestingly, no activity was observed for Tb-FEN1 and T. kodakarensis FEN1 in the presence of Ca 2+ [START_REF] Muzzamal | Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis[END_REF]. Thus, despite their high genetic proximity, the divalent metal ion requirements of Tb-FEN1 and T. kodakarensis FEN1 show more divergences than similarities.

Last, we investigated the effect of salinity on Tb-FEN1 activity. We show that the enzyme activity is inhibited by NaCl, abolishing its activity gradually with increasing NaCl concentrations (Fig. 3D). Specifically, Tb-FEN1 retained 40% cleavage activity at 200 mM NaCl, and only less than 20% cleavage at 400 mM or above. This is similar to what is observed for the M. jannaschii FEN1 [START_REF] Bae | Expression, purification, characterization, and crystallization of flap endonuclease-1 from Methanococcus jannaschii[END_REF], to the exception that Tb-FEN1 still retains ca. 13% relative activity at 600 mM NaCl, while M. jannaschii FEN1 has no detectable activity at and above 400 mM NaCl [START_REF] Bae | Expression, purification, characterization, and crystallization of flap endonuclease-1 from Methanococcus jannaschii[END_REF], suggesting that Tb-FEN1 is slightly more salt-tolerant than M. jannaschii FEN1. The difference in salt tolerance between these two FEN1 homologs cannot be related to the growth optima of the two strains, since both are marine hyperthermophilic isolates with optimal growth salinities ca. 3%, but it may be related to their central metabolism since M. yannaschii is a methanogen and T. barophilus is a peptidotrophe.

Thermostability mechanism assays

As shown in Fig. 1, we found that residues N30, I39, G153, A157, Q172, Y174, N187, L226, I227 and N238 in Tb-FEN1 are conserved in other FEN1 proteins from HA. In contrast, the corresponding residues in eukaryotic FEN1 proteins are the conserved methionine, valine, alanine, cysteine, glutamate, methionine, histidine, phenylalanine, valine, and cysteine (Fig. S1), respectively. To probe a link between these residues and the thermostability of Tb-FEN1, we mutated six residues in Tb-FEN1 back to the corresponding eukaryotic residue by site-directed mutagenesis: N30M, Q172E, Y174M, N187H, L226F and N238C, and purified these six mutant proteins (Fig. S2). We then tested their cleavage activity and thermostability. As shown in Fig. S3, we found that above five mutants displayed the wild-type 5'-flap DNA cleavage activity except for the N238C mutant. As shown in Fig. S4, the N30M, Q172E, Y174M, and N187H mutants exhibited wild-type thermostability. However, the L266F mutant displayed only 87% activity relative to the wild-type protein, suggesting that the L266 residue might play a role in the thermostability of Tb-FEN1.

Mutational analyses of Tb-FEN1

Tb-FEN1 possesses all conserved motifs present in FEN1 homologs from Archaea and eukaryotes, suggesting that this FEN1 resembles other archaeal and eukaryotic FEN1 proteins despite the biochemical specificities we demonstrated above. The human FEN1 is a well-characterized FEN1, and the solved crystal structure of the enzyme with the 5′-flap DNA demonstrates the amino acid residues of the human FEN1 that surround the flap (Fig. 4A) [START_REF] Song | Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage[END_REF]. For clarity, the corresponding residues in Tb-FEN1 are shown in parentheses (Fig. 4A). To clarify the function of these amino acid residues in Tb-FEN1, we have mutated five of the 9 conserved residues in the enzyme that are in the conserved motifs present in other FEN1 homologs.

By using site-directed mutagenesis, we successfully engineered the Tb-FEN1 K87A, R94A, E154A, R186A and D236A mutants. These mutant proteins were expressed and purified as described for the wild-type protein (Fig. 4B).

The cleavage efficiencies of the Tb-FEN1 K87A, R94A, E154A, R186A and D236A mutants we determined by performing DNA cleavage reactions using 5′-flap DNA as a substrate. The Tb-FEN1 mutants K87A, R94A and E154A displayed no cleavage activity at all the tested protein concentrations (Figs. 5A, 5B and 5C), suggesting that the mutations of K87, R94 and E154 to alanine lead to the complete loss of activity. The Tb-FEN1 R186A mutant displayed wild-type cleavage efficiency (Fig. 5D), thereby indicating that this substitution to alanine has no detectable impact on enzyme activity. The Tb-FEN1 D236A mutant was the only mutant displaying reduced cleavage efficiencies (Fig. 5E) in comparison to the wild-type protein, emonstrating that the enzyme activity is only partially inhibited by the mutation of D236 to alanine. Thus, residue D236 is important for the enzyme to reach its full activity, but not essential for catalysis. Overall, our data suggest that residues K87, R94, and E154 in Tb-FEN1 are essential for cleaving 5′-flap DNA.

DNA-binding of the Wild-type/Mutant Tb-FEN1

To test whether the wild-type/mutant Tb-FEN1 proteins can bind 5′-flap DNA, we performed DNA binding assays by incubating the wild-type/mutant enzymes with the 5′-flap DNA substrate. As shown in Fig. 6A, the 5′-flap DNA substrate was gradually bound with increasing wild-type protein concentrations. At 2.4 μM, binding reached 89%, thereby suggesting that Tb-FEN1 can bind effectively 5′-flap DNA.

In comparison, the K87A, R94A, E154A and D236A mutants displayed reduced binding efficiencies at all tested concentrations (Figs. 6B, 6C, 6D and 6F), demonstrating that residues K87, R94, E154 and D236 in Tb-FEN1 are involved in DNA substrate recognition and binding. The Tb-FEN1 R186A mutant had the lowest binding efficiency at 0.6 μM and 1.2 μM (Fig. 6E). However, the Tb-FEN1 R186A mutant displayed wild-type binding at 2.4 μM. Thus, it is probable that residue R186 is to involved directly in 5′-flap DNA substrate recognition but may participate in the proper folding of this site.

Kinetic analysis of Tb-FEN1

Under single-turnover condition where the enzyme concentration was ten-fold 2, demonstrating that the kendo values increased as the reaction temperatures increased.

Discussion

Genomic analyses have shown that FEN1 is present in Eukarya and Archaea, and possesses an endonuclease/5′-3′ exonuclease activity that can remove 5′-flap from DNA.

In the present work, we provide the biochemical characteristics and catalytic mechanism of the FEN1 from the hyperthermophilic euryarchaeon T. barophilus Ch5, demonstrating that Tb-FEN1 is indeed capable of effective excision of 5′-flap from DNA. However, the Tb-FEN1 exhibits several biochemical characteristics distinct from the reported FEN1 homologs from other HA.

Tb-FEN1 has no activity on pseudo Y DNA

In addition to removing 5′-flap from DNA, FEN1 can normally cleave efficiently pseudo Y DNA [START_REF] Rao | Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements[END_REF]. However, to our surprise we found that Tb-FEN1 shows no activity towards the pseudo Y DNA at whatever concentration of the protein and substrate tested, which sharply contrasts with other FEN1 homologs from eukaryotes or other Archaea, even those from the genetically closely related species P. horikoshii and P. furiosus [START_REF] Matsui | Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically[END_REF]Hosfield et al., 1998a). Since Tb-FEN1 possesses 85% amino acid identity with P. furiosus FEN1, a question arose: why the former cannot cleave pseudo Y DNA but the latter can. The sequence comparison of the amino acid sequences of Tb-FEN1 and other archaeal and eukaryotic FEN1 proteins shows that several differences might explain this divergence. Residue T126 in Tb-FEN1 is distinct from the conserved lysine or arginine in other archaeal and eukaryotic FEN1 proteins (Fig. 1A). Additionally, residue H159 in Tb-FEN1 is also different from the conserved tyrosine in archaeal FEN1 and alanine or glutamate in eukaryotic FEN1 (Fig. 1A). These residue differences might alter the ability of Tb-FEN1 to cleave pseudo Y DNA.

Tb-FEN1 has strong thermostability

Strong thermostability is a unique characteristic of proteins from hyperthermophiles. As expected since it originates from the hyperthermophilic euryarchaeon T. barophilus Ch5, Tb-FEN1 is quite thermostable. To our knowledge, it is the most thermostable among the reported FEN1 proteins, since it still retains activity after 20 min. at 100°C. Likewise, the FEN1 homolog from the hyperthermophilic archaeon M. jannaschii retained activity only up to 95°C for 15 min [START_REF] Rao | Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements[END_REF].

We probed the origin of the thermostability of Tb-FEN1, by substituting the amino acid composition of this endonuclease back to the non-thermostable human FEN1 (summarized in Tables S3). Recent studies have shown that thermophilic proteins have a higher percentage of hydrophobic amino acids than mesophilic ones [START_REF] Panja | Protein thermostability is owing to their preferences to non-polar smaller volume amino acids, variations in residual physicochemical properties and more salt-bridges[END_REF]. As shown in Table S3, the percentage (42.4%) of the hydrophobic amino acid (M, F, A, I, L, V, W and P) in Tb-FEN1 is higher than that in human FEN1 (39.6%).

As claimed by [START_REF] Gamage | Applicability of instability index for in vitro protein stability prediction[END_REF], the proteins with the dipeptide composition-based Instability Index (II) value below 40 are stable proteins. Based on the ExPASy (Expert Protein Analysis System), the II values of Tb-FEN1 and human FEN1 are computed to be 33.74 and 50.09, respectively, which further confirms that Tb-FEN1 should be more thermostable than the human FEN1. Additionally, the hyperthermophilic proteins possess an increased percentage of glutamate (E) and lysine (K) and a decreased percentage of glutamine (Q) and histidine (H), so that the E+K/Q+H ratio is >4.5 [START_REF] Farias | Preferred amino acids and thermostability[END_REF]. As expected, the E+K/Q+H ratio (5.34) in Tb-FEN1 is higher than that (2.7) in human FEN1.

In addition to amino acid composition, the conserved residues in FEN1 proteins from HA that are not conserved in eukaryotic FEN1 homologs might be a determinant for strong thermostability of hyperthermophilic archaeal FEN1 proteins. Such ten residues are present in hyperthermophilic archaeal FEN1 proteins (Fig. S1). Intriguingly, we revealed that only residue L266 in Tb-FEN1 seems to be partially responsible for the strong thermostability exhibited by the Tb-FEN1 protein. Considering that only a low reduction in activity (13%) was observed in the heated L266F mutant relative to the heated wild-type protein, we proposed that a network interaction of residue L266 and the other nine residues in hyperthermophilic archaeal FEN1 proteins might contribute to its thermostability.

Roles of the conserved residues in Tb-FEN1

Human FEN1 is a well-characterized structure-specific endonuclease. Residue D233 in human is essential for DNA substrate cleavage and binding [START_REF] Shen | Essential amino acids for substrate binding and catalysis of human flap endonuclease 1[END_REF].

The corresponding residue D236 in P. furiosus FEN1 plays an important role in divalent metal ion binding [START_REF] Allawi | Modeling of flap endonuclease interactions with DNA substrate[END_REF]. In this work, our mutational studies showed that the mutation of residue D236 in Tb-FEN1 to alanine leads to the partial loss of the catalytic activity and reduced binding efficiency, thereby confirming the role of the highly conserved aspartate residue in FEN1 homologs.

Residue E160 in human FEN1 is essential for DNA substrate cleavage and binding via Mg 2+ coordination [START_REF] Shen | Functional analysis of point mutations in human flap endonuclease-1 active site[END_REF]. In addition, the corresponding residue E154 in P. furiosus FEN1 may bridge the two metal ions through water-mediated contacts [START_REF] Allawi | Modeling of flap endonuclease interactions with DNA substrate[END_REF]. In this work, we confirmed that this highly conserved residue E154 in Tb-FEN1 is also essential for substrate cleavage and binding.

The mutation R192F in human FEN1 partially abolishes the cleavage activity [START_REF] Song | Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage[END_REF]. In contrast, we found that the Tb-FEN1 R186A mutant retains wildtype activity. This difference might originate from the different amino acid substitutions used (Ala instead of Phe). Our binding data further demonstrate that residue R186 in Tb-FEN1 is possibly not directly involved in substrate recognition and binding.

The invariant basic residues K93 and R100 in human FEN1 form ion pairs with the scissile phosphate to facilitate hydrolysis [START_REF] Tsutakawa | Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily[END_REF]. Similar observations are made for residue R94 in P. furiosus FEN1 [START_REF] Allawi | Modeling of flap endonuclease interactions with DNA substrate[END_REF]. In Tb-FEN1, residues K87 and R94 are analogous to residues K93 and R100 in human FEN1, respectively. In this work, we show that the K87A and R94A mutations lead to the loss of cleavage activity and a reduction in DNA substrate recognition and binding, which further confirms the function of the conserved basic amino acid residues.

Activation energy

In this work, the kendo values for cleaving 5′-flap DNA by Tb-FEN1 were estimated at various temperatures ranging from 25 o C to 45 o C. By using the Arrhenius equation, the log of kendo values was plotted as a function of the reaction temperature to yield an activation energy (Ea) of 35.7 ± 4.3 kcal/mol (Fig. 8), which represents the energy barrier for the removal of 5′-flap from DNA. Under single-turnover conditions, other FEN homologs have been suggested to be rate-limited by conformational change rather than rate-limited by catalytic chemistry of phosphodiester bond hydrolysis [START_REF] Kim | Structural changes measured by Xray scattering from human flap endonuclease-1 complexed with Mg 2+ and flap DNA substrate[END_REF][START_REF] Kim | Human flap endonuclease-1: conformational change upon binding to the flap DNA substrate and location of the Mg 2+ binding site[END_REF][START_REF] Zheng | Distinct roles of two Mg 2+ binding sites in regulation of murine flap endonuclease-1 activities[END_REF][START_REF] Algasaier | DNA and protein requirements for substrate conformational changes necessary for human flap endonuclease-1-catalyzed reaction[END_REF][START_REF] Song | Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage[END_REF].

Therefore, the activation energy measured here could be an indication of the activation barrier for the conformational change rather than hydrolysis. To our knowledge, this is the first report on an activation energy (Ea) for 5′-flap removal from DNA by FEN1.

Conclusion

In summary, we investigated the biochemical characteristics of FEN1 from the hyperthermophilic euryarchaeon T. barophilus Ch5 and revealed the function of five conserved residues of the enzyme. Interestingly, Tb-FEN1 can remove 5′-flap from DNA, displaying maximal activity in the presence of Mg 2+ at pH 7.0-9.5. However, Tb-FEN1 has no cleavage activity on pseudo Y DNA, which sharply contrasts with other eukaryotic and archaeal FEN1 homologs. Importantly, we found that Tb-FEN1 is the most thermostable among the reported FEN1 proteins. Additionally, we estimated for the first time the activation energy of DNA cleavage by Tb-FEN1 (35.7 ± 4.3 kcal/mol), which represents the energy barrier for the removal of 5′-flap from DNA.

Mutational studies demonstrate that residues K87, R94, and E154 in Tb-FEN1 are essential for substrate binding and catalysis. A. The human FEN1-substrate complex (5UM9) shows DNA distortion at the flap junction [START_REF] Song | Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage[END_REF]. This figure was adapted from [START_REF] Song | Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage[END_REF]

Figure legends

  higher than DNA concentration, we performed a time course experiment of DNA cleavage catalyzed by Tb-FEN1 at 45 o C. As shown in Fig. 7A, the 5′-flap DNA substrate was gradually cleaved as the reaction time extended. Almost all the 5′-flap DNA substrate was cleaved by Tb-FEN1 when the reaction time reached 10 min or more, thereby indicating that the enzyme can cleave effectively 5′-flap DNA. The molar amount of the remaining DNA substrate in the DNA cleavage reactions catalyzed by Tb-FEN1 was quantified and plotted against the reaction time and then fitting with a single-exponential decay equation to yield the kendo and A values (Fig. 7B). The kendo and A values were estimated to be 0.42 ± 0.04 min -1 and 89 ± 3 nM, respectively. Similar kinetic experiments of 5′-flap DNA cleavage reactions catalyzed by Tb-FEN1 were performed at 25°C, 30°C, 35°C, and 40°C, respectively. The corresponding results are shown in Figs. S5, S6, S7, and S8 in supplementary data, respectively. The corresponding kendo and A values are summarized in Table

Fig. 1 .

 1 Fig. 1. The genome of T. barophilus Ch5 encodes a putative FEN1. A. Sequence

Fig. 2 .Fig. 3 .

 23 Fig. 2. DNA cleavage by Tb-FEN1. DNA cleavage reactions were performed with 300,

Fig. 4 .

 4 Fig. 4. Potentially key amino acid residues of Tb-FEN1 that surround the 5′-flap.

  . The 5′flap DNA is colored with orange and the residues are labeled with sticks. Residues D34 and D86 coordinate metal ion B and Residues D181 and D233 coordinate metal ion A. The corresponding amino acids of Tb-FEN1 are shown in the parentheses. B.Purification of the Tb-FEN1 K87A, R94A, E154A, R186A, and D236A mutants.

Fig. 5 .

 5 Fig. 5. Cleavage of 5′-flap DNA catalyzed by the Tb-FEN1 mutants. The 5′-flap

Fig. 6 .

 6 Fig. 6. DNA-binding by the wild-type/mutant Tb-FEN1. The 5′-flap DNA was

Fig. 7 .

 7 Fig. 7. Kinetic analysis of DNA cleavage catalyzed by Tb-FEN1 at 45°C. DNA

Fig. 8 .

 8 Fig. 8. Activation energy for DNA cleavage catalyzed by Tb-FEN1. Ln (kendo) was

Table 1

 1 Sequences of the oligonucleotides used to clone the Tb-FEN1 gene and construct its mutantsThe italic bases represent restriction sites.

	The substitution bases are underlined.

Table 2

 2 Kinetic constants for cleaving 5′-flap DNA by Tb-FEN1 at varied temperatures

	Temperature ( o C)	kendo (min -1 )	A (nM)
	25	0.0079 ± 0.005	80 ± 1
	30	0.029 ± 0.002	93 ± 3
	35	0.072 ± 0.01	86 ± 5
	40	0.13 ± 0.02	80 ± 3
	45	0.42 ± 0.04	89 ± 3

Acknowledgements

We thank Prof. Yulong Shen at Shandong University for kindly providing the E. coli BL21 (DE3) RIL strains and Prof. Yong Gong at Institute of High Energy Physics, Chinese Academy of Sciences, China for kindly providing the pET-28at-plus vector.

Funding

This work was supported by the Provincial Natural Science Foundation of Jiangsu Province (No. BK20191219), High Level Talent Support Program of Yangzhou University and the Academic Leader of Middle and Young People of Yangzhou University Grant to LZ, and the Open Project of Jiangsu Key Laboratory of Marine Bioresources and Environments (No. SH20201206), Jiangsu Ocean University, China to CJ.

Author contributions

LZ and PO designed experiments; TL, DJ, LW, KC, LL, and ZL performed experiments; LZ, TL, WL, DJ, CJ and PO analyzed data; LZ and PO wrote and revised the paper.

Declaration of Interest Statement

All authors declare that there is no conflict of interests regarding the publication of this paper.

Supplementary materials

Supplementary materials for this article are provided.


Amélie Veillere
email: veillere@icmcb-bordeaux.cnrs.fr

Jean-Marc Heintz

Namas Chandra

Joël Douin

Michel Lahaye

Grégory Lalet

Cécile Vincent

Jean-François Silvain

A Veille `re

J.-M Heintz

J.-F Silvain

Influence of the interface structure on the thermo-mechanical properties of Cu-X (X = Cr or B)/carbon fiber composites

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Alternatively, the use of CF as reinforcement in copper matrices offer a good compromise between thermo-mechanical properties In the field of power electronics, the size of the electronic devices is continually decreasing as their number are increasing, resulting in higher power and heat dissipated by the package. In the past 40-50 years, different types of metal matrix composites (MMCs), especially with aluminum and copper matrices, have been investigated to try to solve this problem of thermal management of Silicon chips [1][2][3]. It is necessary to dissipate the excess thermal energy in order to prevent heating and consequent deterioration of the Silicon chip. The material system of choice should not only have a very good thermal dissipation, but high reliability. The latter is greatly limited by the critical difference between the coefficients of and high conductivity, as well as offering a lower density than copper and good machinability. In a composite, general mechanical and/or physical properties are linked to the properties of the matrix, the reinforcement and the interfacial zone. The major problem in the development of the Cu/CF composites is the absence of chemical reaction between copper and carbon. Indeed, after several thermal cyclings the initial mechanical bonding (induced by the CTE mismatch between copper and carbon, after cooling) is degraded, thus leading to a weak interface and a bad transfer of properties between the matrix and the reinforcement [5][6][7][8][9]. To solve this problem, an alloying element such as chromium or boron is added to the copper matrix in order to confer, after diffusion, a strong chemical interface. This paper presents a detailed characterization of such interfaces, in terms of chemical and microstructural analyses, and establishes a link between these interfacial properties and the thermo-mechanical properties of the MMCs. thermal expansion (CTE) of the heat sink (generally in copper, 17 Â 10À K À1 ), and of the ceramic substrate (mainly alumina, ) [4]. More recently, MMCs made with copper reinforced by diamond particles have been investigated to replace the bulk copper heat sink [5][6][7]. These kinds of MMCs improve the thermo-mechanical properties compared to those of copper, but to achieve these results 60 vol.% of diamond was added in the matrix, and such amount implies a very difficult machinability of the composite for industrial applications.

Experimental methods

Materials

Chromium and boron have been selected as additive elements to the copper matrix because they react with carbon to form

A B S T R A C T
This study focuses on the fabrication, for power electronics applications, of adaptive heat sink material using copper alloys/carbon fibers (CF) composites. In order to obtain composite material with good thermal conductivity and a coefficient of thermal expansion close to the ceramic substrate, it is necessary to have a strong matrix/reinforcement bond. Since there is no reaction between copper and carbon, a carbide element (chromium or boron) is added to the copper matrix to create a strong chemical bond. Composite materials (Cu-B/CF and Cu-Cr/CF) have been produced by a powder metallurgy process followed by an annealing treatment in order to create the carbide at the interphase. Chemical (Electron Probe Micro-Analysis, Auger Electron Spectroscopy) and microstructural (Scanning and Transmission Electron Microscopies) techniques were used to study the location of the alloying element and the carbide formation before and after diffusion. Finally, the thermo-mechanical properties have been measured and a promising composite material with a coefficient of thermal expansion 25% lower than a classic copper/carbon heat sink has been obtained. carbide compounds (negative Gibbs energy) [START_REF] Veille `re | Drains thermiques adaptatifs: Cuivre allie ´/Fibre de Carbone[END_REF]. The copperbased alloys are Cu-B (0.05 wt.% of B) and Cu-Cr (0.63 wt.% of Cr). The amounts of boron and chromium have been chosen in order to have the alloying element in solid solution in copper and prevent the formation of particles inside the matrix, which can decrease the thermal properties. The powders are prepared by CERAM (United Kingdom), by an atomization process, with a particle size under 25 mm. Two kinds of chopped CF, manufactured by Nippon Graphite Fiber Corporation, are investigated. Both have a diameter of 10 mm, a length ranging from 100 to 400 mm, and a CTE of À1.0 Â 10 À6 K À1 and 12 Â 10 À6 K À1 along and perpendicular to their axis, respectively. The difference between these two kinds of CF is their thermal conductivity along their axis which is about 320 W m À1 K À1 and 900 W m À1 K À1 for the CN80C and XN100, respectively (Table 1). It has to be mentioned that the thermal conductivity measured perpendicular to the fiber axis is equivalent for both fiber and in the order of 5 W m À1 K À1 .

Copper alloy powders are mechanically mixed with CF (volume fraction equal to 30%) and are then uniaxially hot pressed at 950 8C under 50 MPa for 20 min to obtain dense materials. During the sintering cycle, a reducing atmosphere of argon/hydrogen (5 vol.%) is used to first deoxidize the initial Cu alloys powder and then prevent the oxidation of the copper during densification. An optimized annealing treatment is then performed (1000 8C, 24 h, under reducing atmosphere) on the most promising composite (Cu-Cr/CN80C) to increase the diffusion of the alloying element (Cr) toward the Cu/CF interface and therefore create stronger carbide interface bonds.

Chemical and microstructural characterizations

After densification, the samples were analyzed in cross and parallel section using an optical microscope (Olympus PMG3). Matrix, interface and reinforcement microstructures were analyzed using scanning electron microscopy (SEM, JEOL 6700F). In order to investigate the microstructure of the interface at a nanometric scale, transmission electron microscopy was also used (TEM, JEOL 200CX).

The chemical distribution of the alloying element inside the composite was analyzed using Electron Probe Micro-Analyses (EPMA, CAMECA SX 100) equipped with wavelength dispersive spectrometry (WDS); the accelerating voltage was 20 kV. Auger Electron Spectroscopy (AES, VG Microlab 310F) was used in order to analyze the chemical composition of the matrix, reinforcement and interfaces at a nanometric scale (analyses diameter around 30 nm). The AES analyses were made with an acceleration voltage of 10 keV and a beam current of 4 nA. The energy ranges investigated in this technique were the following for each element: 890-950 eV (Cu LM2 ), 520-600 eV (Cr LM2 ) and 250-275 eV (C KL1 ). The peaks in the energy spectrum were correlated to an AES database [START_REF] Childs | Handbook of Auger Electron Spectroscopy[END_REF] or peaks from reference compounds obtained from the same equipment to identify specific elements. Data acquired along the line profile were computer-analyzed using a non-linear least square fitting (NLLSF). Finally the atomic concentration of each of the identified elements is plotted as a function of distance from a reference point.

Thermo-mechanical measurements

The thermo-mechanical properties have been measured on each composite material. The thermal diffusivity (a) was measured using a flash laser technique (Netzsch LFA 457). In this technique, a pulse laser excites the bottom-surface of the sample and the variation of temperature is measured in the opposite face. This measure is performed on 6 mm diameter samples and between 2 and 5 mm thick. The faces must be parallel in order to obtain accurate and reproducible results. Before the experiment, the sample is covered with a thin film of graphite to avoid radiation. The thermal conductivity (l) of the material is deduced using the relation l = aÁrÁCp, where r correspond to the density of the composite measured by the Archimedes method and Cp is the specific heat at constant pressure calculated with a rule of mixture.

The coefficient of thermal expansion (CTE) was measured with a differential dilatometer (Netzsch LFA 457). The heating rate was 2 8C/min and ranged from room temperature to 250 8C under argon atmosphere. The CTE value is an average value measured between 100 8C and 200 8C.

Results and discussion

Densification

The final density of the composite material is close to 97% and no specific degradation of the CF was observed. The optical micrographs of the samples show a strong anisotropic structure (Fig. 1), with an orientation of the CF perpendicular to the pressure direction. This anisotropy is due to the strains and stresses imposed by the pressing conditions and geometry, and will be reflected in the anisotropic thermal (l) and thermo-mechanical (CTE) properties of the composite.

Microstructure and interface chemistry

Fig. 2a shows a back-scattered SEM micrograph of the Cu-Cr/CF sample before annealing treatment. In this micrograph, the carbon fibers appear in black, the Cu in light gray and the Cr in dark gray. We can observe Cr clusters on the Cu matrix and some at the Cu/CF interfaces. These observations are confirmed by the WDS-EPMA mapping of the Cr in a representative area of the sample (Fig. 2b). The high temperature of the densification process (950 8C) allows partial diffusion of the alloying element toward the interface.

Fig. 3 shows a back-scattered SEM micrograph (Fig. 3a) and the chromium WDS-EPMA mapping (Fig. 3b) of the Cu-Cr/CF sample after an annealing treatment at 1000 8C for 24 h. Both analyses show that most of the Cr has migrated toward the Cu/CF interface and the Cu matrix is almost free of Cr. At this scale of analysis, the alloying element does not appear homogeneously dispersed at the interface, as portions seem without alloying element. The nonuniformity and discontinuity of the interphase could be linked to the inhomogeneous dispersion of the alloying element in the initial copper alloy powders. Fig. 4 shows an EPMA mapping of the Cr in the initial Cu-Cr powder where it is clear that the Cr (in white) is not distributed evenly in the copper grains. This heterogeneity induces a non-uniform diffusion of the alloying element in the copper matrix and may explain the thickness variation of the Cu/CF interfacial zone. We can also observe, circled in Fig. 3b,a Both of these chemical analyses confirm the diffusion, after annealing treatment, of the alloying element through the Cu matrix toward the Cu/CF interface, with the formation of a carbide.

Interface morphology and crystallographic structure

TEM studies have been performed on both Cu-Cr/CF and Cu-B/ CF composite materials, in order to analyze the interface at a nanometric scale and determine its crystallographic structure. The thickness of the carbide interphase has been measured in both cases, ranging from 50 nm to 1 mm. We also notice its presence all around the fiber, contrary to the first SEM observations. Fig. 6 shows TEM micrographs and diffraction patterns obtained on the Cu-B/CF composite. Micrograph 6a shows 3 distinct areas: the matrix, the interfacial zone and the CF, from top to bottom. In this micrograph, the CF is not attached to the interfacial zone, this phenomenon is an artifact due to the thin film preparation for the TEM analyses. The diffraction pattern acquired in the Cu matrix shows the presence of metallic Cu with no trace of alloying element in cluster shape. Therefore if an alloying element was present in the matrix, it would be in a small amount and in a solid solution with Cu. The diffraction pattern of the CF (Fig. 6d) is composed of rings formed by its graphitic plane structure, and of a systematic diffraction line {1 1 1} due to the preferential orientation of the graphitic planes along the fiber axis. A typical diffraction pattern obtained at the interphase area reveals a ringlike morphology which is characteristic of polycrystalline grains with sizes much smaller than the diffraction diaphragm used to obtain that pattern (10 mm). The indexation of the rings clearly shows the presence of boron carbide B 4 C (R3 ¯m space group). A zoom on the interfacial zone is shown in Fig. 6e and a dark field of the same zone using the {1 0 4} B 4 C diffraction ring can be seen in Fig. 6f. From these micrographs, we can observe the nanometric size (between 5 and 30 nm) of B 4 C grains (in white). The same analyses have been performed on the Cu-Cr/CF composite material (Fig. 7a-c). Three zones are visible (Fig. 7a andb) and the electronic diffraction analyses (Fig. 7c) of each zone identified the crystallographic structures of the Cu matrix, the CF and the interfacial zone composed of chromium carbide Cr 3 C 2 (Pnma space group).

Thermo-mechanical properties

The thermal conductivity (TC) and the coefficient of thermal expansion (CTE) of each material have been measured. Both TC and CTE depend on the quality of the matrix/reinforcement interface [START_REF] Gwinn | [END_REF]. Two kinds of interfaces can be defined:

-A mechanical interface due to the mechanical clamping arising from CTE mismatch-induced thermal stresses during cooling. In this section, the measured TC and CTE will be related to the matrix, reinforcement and interface type.

Thermal conductivity (TC)

Thermal diffusivity is measured along the pressure direction of the densification process due to technical limitation during the experiments. As shown in Section 3.1, the densification process induces an anisotropic structure in the samples with a preferential orientation of the CF perpendicular to the pressure axis. Therefore the measurement corresponds to the thermal diffusivity perpendicular to the fiber axis. Due to the low transverse TC of the fiber (5 W m À1 K À1 ), it should be noted that we measured the lowest thermal diffusivity of our composite material. Measures were taken for both Cu-Cr/CF and Cu-B/CF composites. The calculated TC from the experimental thermal diffusivities are given in Table 2.

The first point that could be highlighted is the higher TC of the composite materials with the XN100 CF compared to those with the CN80C fibers. However, it should be noticed that both CF have the same TC in transverse direction but a different one along the CF axis (Table 1). The difference of TC measured between these two sets of composite materials could only be explained by an imperfect orientation of the CF inside the matrix. Indeed, if the CF are not perfectly perpendicular to the pressure direction, a parallel contribution on the TC must be taken into account. This phenomenon has already been demonstrated [START_REF] Vincent | Le composite cuivre/nanofibres de carbone[END_REF] in the case of a Cu/carbon nanofibers (CNF) composite material using finite element simulation. The calculated TC perpendicular to the CNF axis increases with the orientation of the CNF, as shown in Fig. 8.

Another observation is the similar TC values of composites with same reinforcement and different matrices (Cu or Cu-Cr or Cu-B), while the initial TC of the pure matrices are different. Indeed, the copper alloy matrices (Cu-Cr and Cu-B) have a lower TC than pure Cu whereas the final composites have the same TC. This phenomenon can be explained by the diffusion of the alloying element at the Cu/CF interfaces which leads to the formation of a carbide at the interface and a matrix almost free of alloying element. This means that the value of the TC depends mostly on the TC of the matrix and reinforcement and not on the interface type (mechanical or chemical).

Coefficient of thermal expansion (CTE)

As mentioned previously, the densification process leads to a random orientation of the CF in a plane perpendicular to the pressure direction. Table 2 shows the CTE variation, inside this plane, for different Cu/CF and Cu-X/CF composites.

3.4.2.1. Several points should be emphasized.

Whatever the matrix used (pure Cu or Cu alloys), the addition of CF inside the matrix leads to a decrease of the CTE of the composite material. For Cu/CF composites, where there is no chemical interface between the matrix and the reinforcement, CTE values are identical for both CF (CN80C and XN100). Therefore the decrease of the CTE is mainly attributed to a composite effect where the mechanical clamping of the CF by the Cu matrix allows a transfer of properties between the matrix and the reinforcement. Indeed, if we assumed that the CF are perfectly oriented in the plane but randomly dispersed, a simple rule of mixture will give an average CTE value of the CF equal to 5.5 Â 10 À6 K À1 and for the composite close to 14 Â 10 À6 K À1 . Our experimental values are in very good agreement with this simple rule of mixture. For Cu-X/CF composites, with Cu-Cr or Cu-B matrices, an improvement of the CTE compared to the Cu/CF reference composite is measured. It should be noted that CTE measured for copper alloy composites with CN80C fibers are slightly lower than those with XN100. Chemical analyses on both CF provide some hypothesis to this small but real effect. XPS analyses on both fibers show a similar behavior where C 1s linked to graphite is mainly obtained. However, Raman analyses on both fibers give different results. Raman spectroscopy is used to measure the crystallinity of the fiber. Indeed, if the CF is perfectly crystalline, only one peak close to 1580 cm À1 , corresponding to the G-band of graphite, appears in the Raman spectrum. When defects are present in the volume of interrogation, another peak called disorder-induced band (D-band) is detected around 1350 cm À1 . The intensity ratio between the G-band and the D-band (I G /I D ) can be linked to the defect quantity on the CF surface. Raman spectroscopy analyses of CN80C and XN100 fibers give values of I G /I D of 4.5 and 7.3, respectively. These ratios confirm the higher quantity of crystalline defects on the CN80C fiber, therefore this type of CF is more reactive if we assume that defects are linked to the nucleation sites for carbide formation. Finally, stronger chemical interfaces are present between copper alloy matrices and CN80C CF than with XN100 CF. These stronger chemical links can be associated to higher transfer properties which result in lower measured CTE.

3.4.2.2. Influence of the annealing treatment. An optimized annealing treatment has been performed (1000 8C, 24 h, under reducing atmosphere) on the composite with the lowest CTE (sample S5, Cu-Cr/CN80C) in order to increase the diffusion of the alloying element (Cr) toward the Cu/CF interface. This diffusion phenomenon is clearly seen in the chemical analyses (Section 3.3) and the formation of chromium carbide is identified. In order to quantify the effect of this chemical interface, the same treatment was performed on a reference sample having only a mechanical interface (sample S2, Cu/CN80C). The CTE of both samples have been measured before and after annealing treatment.

The annealing treatment on the reference sample (sample S2) lead to a deterioration of the thermo-mechanical properties, the CTE value of this sample increasing from 14 Â 10 À6 K À1 to 17 Â 10 À6 K À1 . As explained previously, there is no chemical reaction between Cu and CF therefore the interfacial link is only a mechanical link due to the CTE mismatch between the Cu and the CF. The annealing treatment was made at 1000 8C without any constraint; therefore the dilatation of Cu was much higher than that of the CF and lead to a delamination between the matrix and the reinforcement, and a destruction of the mechanical interface. Therefore pure copper matrix CTE is measured after this heat treatment.

However, in the case of sample S5 where a chemical interface is present, we observe a decrease of the CTE from 12 Â 10 À6 K À1 to 11 Â 10 À6 K À1 . The annealing treatment is advantageous in this case because it leads to the full diffusion of the alloying element toward the interfacial zone and the formation of a stronger carbide chemical bond (Cr 3 C 2 ) at the Cu/CF interface. This chromium carbide interphase preserves the contact between the matrix and the reinforcement and avoids delamination of the material which results in a decrease of the composite material CTE.

These thermo-mechanical analyses show that the addition of an alloying element (chromium) produces thermal conductivity equivalent to Cu/CF composites, but with an improvement of 25% for the CTE.

Conclusions

The chemical and microstructural characterizations of the Cu-X/CF composite materials have shown diffusion of the alloying element (X = Cr or B) in the copper matrix toward the copper/ carbon fiber interface. Analyses at a nanometric scale have clearly identified the crystalline structure of the interfacial zone, i.e. chromium carbide (Cr 3 C 2 ) for the Cu-Cr/CF composites and boron carbide (B 4 C) for the Cu-B/CF. Measurements of thermo-mechanical properties for each composite has demonstrated the influence of the interfacial zone composition and the reinforcement type on the material properties. We have produced a promising composite by adding chromium in the copper matrix and using CN80C carbon fibers as reinforcement. Compared to a similar composite material without alloying element, an improvement of 25% on the coefficient of thermal expansion was measured.

6 8 Â

 8 10À K À1 6

Fig. 1 .

 1 Fig. 1. Optical micrographs of the composite material in (a) cross and (c) parallel section after densification and (b) schematic of the CF orientation.

  Fig.2ashows a back-scattered SEM micrograph of the Cu-Cr/CF sample before annealing treatment. In this micrograph, the carbon fibers appear in black, the Cu in light gray and the Cr in dark gray. We can observe Cr clusters on the Cu matrix and some at the Cu/CF interfaces. These observations are confirmed by the WDS-EPMA mapping of the Cr in a representative area of the sample (Fig.2b). The high temperature of the densification process (950 8C) allows partial diffusion of the alloying element toward the interface.Fig.3shows a back-scattered SEM micrograph (Fig.3a) and the chromium WDS-EPMA mapping (Fig.3b) of the Cu-Cr/CF sample after an annealing treatment at 1000 8C for 24 h. Both analyses show that most of the Cr has migrated toward the Cu/CF interface and the Cu matrix is almost free of Cr. At this scale of analysis, the alloying element does not appear homogeneously dispersed at the interface, as portions seem without alloying element. The nonuniformity and discontinuity of the interphase could be linked to the inhomogeneous dispersion of the alloying element in the initial copper alloy powders. Fig.4shows an EPMA mapping of the Cr in the initial Cu-Cr powder where it is clear that the Cr (in white) is not distributed evenly in the copper grains. This heterogeneity induces a non-uniform diffusion of the alloying element in the copper matrix and may explain the thickness variation of the Cu/CF interfacial zone. We can also observe, circled in Fig.3b, a higher segregation of Cr on the CF tip. This tip segregation is due to the carbon pending bond present in these areas.AES line profiles have been obtained across a Cu/CF interphase. One representative line profile is shown in Fig.5. Atomic concentrations of Cu, C and Cr were measured along the line. A

Fig. 2 .

 2 Fig. 2. Back-scattering SEM micrographs of sample S5 (a) before and (b) after annealing treatment at 1000 8C/24 h under Ar/H 2 (5%) atmosphere.

Fig. 3 .

 3 Fig. 3. Chromium EPMA mapping of the initial Cu-Cr powder.

Fig. 4 .

 4 Fig. 4. EPMA mapping of the chromium in sample S5 (a) before and (b) after annealing treatment.

Fig. 5 .

 5 Fig. 5. AES depth profile through the Cu/CF interface of sample S5 after annealing treatment.

-

  A chemical interface due to the formation of carbide (Cr 3 C 2 or B 4 C) created by the diffusion of the alloying element (Cr or B) at the Cu/CF interface.

Fig. 6 .

 6 Fig. 6. TEM analyses of sample S9. (a) Bright field of the zone with, from top to bottom, the Cu matrix, the interface and the CF; (b) diffraction pattern of the Cu matrix ([0 0 1] copper axis); (c) diffraction pattern of the interfacial zone; (d) diffraction pattern of the CF; (e) zoom on the interfacial zone and (f) dark field of the interfacial zone using the {1 0 4}B 4 C diffraction ring marked on (c).

Fig. 7 .

 7 Fig. 7. TEM micrographs (a) of the Cu-Cr/CF composite material after annealing treatment, (b) magnification of the circled area in (a) and (c) diffraction pattern inside the copper, the interphase and the CF.

Fig. 8 .

 8 Fig. 8. Variation of the thermal conductivity with the orientation of the reinforcement in a Cu/CNF composite material [1].

Table 1

 1 Parameters and thermal properties of the investigated CF.

	Fibers	Diameter (mm)	Length (mm)	Density (g cm À3 )	Thermal	conductivity	CTE (10 À6 K À1 )
					(W m À1 K À1 )
					Axial	Transverse	Axial	Transverse
	CN80C	10	100-400	2.1	320	5	À1.0	12
	XN100	10	100-400	2.1	900	5	À1.0	12

Table 2

 2 Transverse thermal conductivity and parallel CTE of Cu-X/CF composite materials.

	Matrix	Reinforcement	Sample name	Thermal conductivity (W m À1 K À1 )	CTE (10 À6 K À1 )
	Cu	-	S1	400	17
		30 vol.% CN80C	S2	180	14
		30 vol.% XN100	S3	220	14
	Cu-Cr	-	S4	360	17
		30 vol.% CN80C	S5	180	12
		30 vol.% XN100	S6	190	14
	Cu-B	-	S7	370	17
		30 vol.% CN80C	S8	180	12
		30 vol.% XN100	S9	230	13

Acknowledgement

The authors would like to thank the ''Programme inter-re ´gion Midi-Pyre ´ne ´es/Aquitaine, Cuivre allie ´/nanofibre de carbone'' for its financial support.


Gunter Heymann

Birgit Heying

Ute Ch

Rodewald

Bernard Chevalier

Hubert Huppertz

Rainer Po

Á H Huppertz

Á U Ch

Á R Rodewald

Po

High-pressure phases of Tb 2 Ni 2 Sn and Dy 2 Ni 2 Sn

Keywords: Ternary stannides, High-pressure phase, Intermetallics

The W 2 B 2 Co-type ternary stannides Tb 2 Ni 2 Sn and Dy 2 Ni 2 Sn (space group Immm) show pressure-induced reconstructive phase transitions. Under high-pressure (8 GPa) and high-temperature (1,470 K) treatment they transform to the tetragonal Mo 2 B 2 Fe-type high-pressure (HP) modifications: P4/mbm, a = 732.6(1) pm, c = 371.71(9) pm, wR2 = 0.0319, 225 F 2 values for HP-Tb 2 Ni 2 Sn and a = 730.3(2) pm, c = 369.1(2) pm, wR2 = 0.0221, 230 F 2 values for HP-Dy 2 Ni 2 Sn, with 12 variables per refinement. At 8 GPa HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn started to decompose into the equiatomic stannides TbNiSn and DyNiSn and a yet unknown phase. Single-crystal diffractometry revealed the following data: TiNiSi type, Pnma, a = 714.9(1) pm, b = 445.9(1) pm, c = 766.4(1) pm, wR2 = 0.0558, 575 F 2 values, 20 parameters for TbNiSn and a = 710.5(1) pm, b = 445.31(9) pm, c = 765.7(1) pm for DyNiSn. The cell volumes of HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn nicely fit into the Iandelli plot of the whole RE 2 Ni 2 Sn (RE = rare earth) series of compounds. The polyanionic networks [NiSn] and [Ni 2 Sn] of the investigated ternary stannides show covalent Ni-Sn bonding with distorted tetrahedral SnNi 4/4 coordination (259-279 pm Ni-Sn) in the RENiSn compounds and a square-planar SnNi 4/4 arrangement (291-292 pm Ni-Sn) in HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn. The nickel atoms form Ni 2 pairs with Ni-Ni distances of 250 pm.

Introduction

The series of RE 2 Ni 2 Sn ternary stannides shows dimorphism. The larger rare earth (RE) elements Ce, Pr, Nd, Sm, Gd, Tb, Dy, and Y form the orthorhombic W 2 B 2 Co-type structure, space group Immm, whereas the smaller ones with Ho, Er, Tm, Lu, and Sc lead to the tetragonal Mo 2 B 2 Fe structure, space group P4/mbm. The crystal chemical data of this series are summarized in Ref. [1]. The common structural motifs of both structures are nickelcentered trigonal prisms which show different condensation patterns.

The interesting point in such dimorphic series concerns the break in structure type. The latter is mostly a consequence of the size of the RE atom (lanthanoid contraction). At a critical size a given structure type is no longer feasible and there are three ways out: (a) the series of compounds ends with that RE element, (b) the structure shows superstructure formation with a well-defined group-subgroup relation, or (c) a reconstructive phase transition to a different structure type but with the same composition occurs [2]. This last version is the case for the RE 2 Ni 2 Sn series.

At the transition point it is sometimes possible to stabilize metastable phases. The compound with the first RE element of the new structure type might form the previous structure type as a high-temperature phase that can be quenched in the case of a reconstructive phase transition. In contrast, the last representative of a series might form a high-pressure (HP) phase with the other structure type. In the field of intermetallics we succeeded in stabilizing such phases in the series of RENiSn, REPdSn, and REAgSn stannides [3,4]. We have now tested the W 2 B 2 Co-type ternary stannides Tb 2 Ni 2 Sn and Dy 2 Ni 2 Sn under similar high-pressure high-temperature conditions and could stabilize the new high-pressure phases HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn with Mo 2 B 2 Fe-type structure. The synthesis conditions, structure determinations, and crystal chemical details are reported herein.

Results and discussion

Structure refinements

Analyses of the X-ray single-crystal diffractometer data sets showed that both specimens consisted of large domains of the HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn phases and very small domains (around 5 %) of the decomposition products TbNiSn and DyNiSn. Consequently we integrated two separate data sets for each crystal. The TbNiSn and DyNiSn data sets showed primitive orthorhombic lattices (Table 1) and the systematic extinctions were compatible with space group Pnma, in agreement with earlier work on CeNiSn [3]. The positional parameters of the cerium compound were taken as starting values. The HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn data sets showed primitive tetragonal lattices and the systematic extinctions were in accordance with space group P4/mbm, similar to Ho 2 Ni 2 Sn [1]. Consequently we used the positional parameters of Ho 2 Ni 2 Sn as starting values. The four structures were then refined using SHEL-XL-97 [START_REF] Sheldrick | SHELXL-97, Program for crystal structure refinement[END_REF]6] (full-matrix least-squares on F 2 ) with anisotropic displacement parameters for all atoms. The lower quality of the DyNiSn data set (the domain size was very small; this led to weak overall intensities) allowed only for an anisotropic refinement of the Dy atoms. Nevertheless, we could prove its TiNiSi-type structure, but with bad residuals. These data are not listed here, but were deposited in a national crystallography database (vide infra).

Refinements of the occupancy parameters gave no hint for deviations from the ideal compositions. The refinements went smoothly to the values listed in Table 2. The final difference-Fourier syntheses revealed no significant residues. The refined atomic positions, isotropic displacement parameters, and interatomic distances are given in Tables 3 and4, respectively. The high-pressure synthesis experiments indicated decomposition of the HP-RE 2 Ni 2 Sn phases towards higher pressures. Indeed, the terbium-containing sample already showed a considerable amount of equiatomic TbNiSn in the Guinier powder pattern and we were able to refine the lattice parameters (Table 1). Only traces of DyNiSn have been observed for the HP-Dy 2 Ni 2 Sn sample and the lattice 4). HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn belong to the large family of compounds with ordered U 3 Si 2 -type structure (Mo 2 B 2 Fetype) and they are the first high-pressure representatives. The crystal chemistry and the bonding peculiarities of these intermetallics have been reviewed [START_REF] Lukachuk | [END_REF]. For further details we refer to this write-up as well as to the work on the normal-pressure NP-RE 2 Ni 2 Sn phases [1]. The Tb atoms are coordinated to two puckered and tilted Ni 3 Sn 3 hexagons. This motif resembles the well-known AlB 2 -type and consequently we can describe TbNiSn as an orthorhombically distorted ordering variant of AlB 2 [13]. More detailed crystal chemical discussion on TiNiSi-type intermetallics is available elsewhere [13][14][START_REF] Parthe | TYPIX-standardized data and crystal chemical characterization of inorganic structure types[END_REF]. The Tb atoms bind to the three-dimensional [NiSn] network via Tb-Ni contacts with Tb-Ni distances of 302 and 307 pm, longer than in HP-Tb 2 Ni 2 Sn. We can thus conclude that stronger Tb-Ni bonding exists in HP-Tb 2 Ni 2 Sn but stronger Ni-Sn bonding exists in TbNiSn. The same holds true for the isotypic dysprosium stannides.

Crystal chemistry

High

Experimental

Synthesis of precursor compounds

Starting materials for the synthesis of the precursor ternary stannides Tb 2 Ni 2 Sn and Dy 2 Ni 2 Sn were pieces of terbium and dysprosium (Smart Elements), nickel wire (Alfa Aesar, [ 1 mm), and tin granules (Merck,[4 mm), all with stated purities better than 99.9 %. The elements were weighed in the ideal 2:2:1 atomic ratio and first arc-melted [16] under an argon pressure of ca. 700 mbar. The buttons were remelted twice to ensure homogeneity. These arc-melted samples contained small amounts of an impurity phase. They were subsequently sealed in evacuated silica ampoules and annealed at 1,070 K for 1 week. The polycrystalline products are stable in air over months.

High-pressure high-temperature treatment

To obtain the tetragonal Mo 2 B 2 Fe-type Tb 2 Ni 2 Sn and Dy 2 Ni 2 Sn compounds, cylindrical BN crucibles (BNP GmbH, HeBoSint Ò P100, Germany) with a fitting BN plate were filled with the carefully milled precursor compounds. The high-pressure and high-temperature experiments took place in a modified Walker-type module in combination with a 1,000-ton press (both devices from the Voggenreiter, Mainleus, Germany). As pressure medium, precast MgO octahedra (Ceramic Substrates & Components, Isle of Wight, UK) with edge lengths of 18 mm were applied. Eight WC cubes (ha 7 % Co, Hawedia, Marklkofen, Germany) with truncation edge lengths of 11 mm compressed the octahedra. Further information on the construction of the assemblies is given in Ref. [17]. parameters listed in Table 1 rely on the single-crystal diffractometer data. Since both single crystals showed tiny secondary domains, we could also refine the structures of TbNiSn and DyNiSn. So far, these two stannides had only been studied on the basis of powder X-ray diffraction [2,[7][8][9].

For comparison with HP-Tb 2 Ni 2 Sn we discuss the TbNiSn structure. Its TiNiSi-type unit cell is presented in The samples were compressed up to 8 GPa in 3.5 h, then heated to 1,470 K within 15 min and kept there for another 30 min. Afterwards, the samples were cooled down to 1,370 K within 30 min, followed by a tempering period of 120 min with an accompanied decrease of temperature down to 1,170 K. Finally, the samples were quenched to room temperature by switching off the heating. The decompression of the assembly required 10.5 h. The recovered MgO octahedra were broken apart and the samples were carefully separated from the surrounding BN crucible. Polycrystalline samples are gray with metallic lustre and stable in air over months.

Scanning electron microscopy

The HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn single crystals investigated on the diffractometers were analyzed using a Zeiss EVO Ò MA10 scanning electron microscope in variable pressure mode with TbF 3 , DyF 3 , Ni, and Sn as standards. No impurity elements heavier than sodium (detection limit of the instrument) were observed. The experimentally determined compositions were close to the ideal ones. The extremely irregularly shaped crystal surfaces hampered determination of absolute values.

X-ray diffraction data

The normal-pressure (NP) and high-pressure (HP) RE 2 Ni 2 Sn samples were characterized by powder X-ray diffraction on a Guinier camera (image plate system, Fujifilm, BAS-1800, Cu Ka 1 radiation and a-quartz (a = 491.30 pm, c = 540.46 pm) as an internal standard). The lattice parameters (Table 1) were obtained from leastsquares refinements. The correct indexing of the patterns was confirmed by intensity calculations [18].

Single crystals of HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn were selected from the HP-HT treated samples by mechanical fragmentation. They were glued to thin quartz fibers using bees wax and their quality was checked by Laue photographs on a Buerger camera (white molybdenum radiation, image plate technique, Fujifilm, BAS-1800). The HP-Tb 2 Ni 2 Sn data set was collected at room temperature by use of a Stoe IPDS-II image plate system (graphite monochromatized Mo radiation; k = 71.073 pm) in oscillation mode. The HP-Dy 2 Ni 2 Sn crystal was measured on a Stoe StadiVari equipped with a Mo microfocus source and a Pilatus 100 K detector with a hybrid-pixel-sensor. Numerical absorption corrections were applied to the data sets. All relevant crystallographic data and details of the data collections and evaluations are listed in Table 2.

Further information on the structure refinements is available. Details may be obtained from Fachinformationszentrum Karlsruhe, D-76344 Eggenstein-Leopoldshafen (Germany), by quoting the registry nos. CSD-427285 (TbNiSn), CSD-427284 (DyNiSn), CSD-427282 (Tb 2 Ni 2 Sn), and CSD-427283 (Dy 2 Ni 2 Sn).

Fig. 1

 1 Fig. 1 Cell volumes of normal-pressure (NP) and high-pressure (HP) phases in the series of RE 2 Ni 2 Sn ternary stannides

Fig. 1

 1 we present the course of the cell volumes within the RE 2 Ni 2 Sn series. HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn nicely extend the tetragonal RE 2 Ni 2 Sn (NP-RE 2 Ni 2 Sn, P4/mbm) ternary stannides towards the larger rare earth elements. The cell volumes are in agreement with the lanthanoid contraction. Both high-pressure phases show slightly smaller cell volumes. Nevertheless, direct comparison of absolute values is difficult, because the structure type changes as a consequence of the reconstructive phase transition. A projection of the HP-Tb 2 Ni 2 Sn structure is presented in Fig. 2. It is best described (from a geometrical point of view) as an intergrowth variant of slightly distorted AlB 2 and CsCl related slabs of compositions TbNi 2 and TbSn. Together, the Ni 2 pairs and the Sn atoms form planar [Ni 2 Sn] networks with distances of 250 pm for Ni-Ni and 292 pm for Ni-Sn, leading to SnNi 4/4 square-planar coordination for the Sn atoms. The Ni-Sn contacts are only of weakly bonding character because they are much longer than the sum of the covalent radii of 255 pm [10]. Significant bonding contributions arise from Tb-Ni with comparatively short distances of 281 and 289 pm (Table

Fig. 2

 2 Fig. 2 Crystal structures of HP-Tb 2 Ni 2 Sn (Mo 2 B 2 Fe-type) and TbNiSn (TiNiSi-type)

Fig. 2 .

 2 The Ni and Sn atoms form puckered Ni 3 Sn 3 hexagons with distances of 259 and 264 pm for Ni-Sn, slightly longer than the sum of the covalent radii. Between the layers the Ni-Sn distances of 279 pm are even longer and thus the inter-layer Ni-Sn interactions are weaker. Such transition metal-tin distances typically occur in shandite

Table 1

 1 -pressure and high-temperature experiments on W 2 B 2 Co-type Tb 2 Ni 2 Sn and Dy 2 Ni 2 Sn led to reconstructive phase transitions to HP-Tb 2 Ni 2 Sn and HP-Dy 2 Ni 2 Sn Lattice parameters of TbNiSn, DyNiSn, HP-Tb 2 Ni 2 Sn, and HP-Dy 2 Ni 2 Sn

	Compound	a/pm	b/pm	c/pm	V/nm 3 Reference
	TbNiSn	714.9(1) 445.9(1) 766.4(1) 0.2443 This work
	TbNiSn	714.6	444.8	766.1	0.2435 [7]
	TbNiSn	713.8(3) 445.0(2) 766.0(3)	0.2433 [8]
	TbNiSn	702.2	442.5	762.6	0.2370 [9]
	DyNiSn*	710.5(1) 445.31(9) 765.7(1) 0.2423 This work
	DyNiSn	711.2	443.9	765.6	0.2417 [7]
	DyNiSn	712.1	445.6	766.0	0.2431 [2]
	DyNiSn	708.8(2) 443.5(2) 765.5(3)	0.2406 [8]

HP-Tb 2 Ni 2 Sn 732.6(1) a 371.71(9) 0.1995 This work HP-Dy 2 Ni 2 Sn 730.3(2) a 369.1(2) 0.1968 This work * Single-crystal diffractometer data

Table 2

 2 Crystal data and structure refinement results for HP-Tb 2 Ni 2 Sn, TbNiSn, and HP-Dy 2 Ni 2 Sn

	Compound	HP-Tb 2 Ni 2 Sn TbNiSn	HP-Dy 2 Ni 2 Sn
	Structure type	Mo 2 B 2 Fe	TiNiSi	Mo 2 B

2 Fe

Unit cell dimensions Table

1

Table 1 Table 1

 11

	Space group	P4/mbm	Pnma	P4/mbm
	Z	2	4	2
	Molar mass/g mol -1 553.95	336.32	561.11
	Calculated density/	9.22	9.14	9.47
	g cm -3
	Absorption	50.2	46.0	52.9
	coefficient/mm -1
	F(000)/e	472	572	476
	Crystal size/lm 3	20 9 30 9 60 20 9 30 9 60 10 9 20 9 30
	Transm. ratio	0.101/0.475	0.293/0.658	0.191/0.600
	(min/max)
	h range/°3-33	3-35	3-33
	Range in hkl	±11, ±11, ±5 ±11, ±6, ±12 ±11, ±11, ±5
	Total no. reflections 2,553	2,901	2,393
	Independent	225/0.1394	575/0.0503	230/0.0397
	reflections/R int
	Reflections with	223/0.0530	492/0.0274	212/0.0186
	I C 2r(I)/R r
	Data/parameters	225/12	575/20	230/12
	Goodness-of-fit on	0.902	1.117	1.001
	F 2
	R1/wR2 for	0.0403/0.0310 0.0241/0.0511 0.0125/0.0219
	[I C 2r(I)]
	R1/wR2 for all data 0.0474/0.0319 0.0326/0.0558 0.0143/0.0221
	Extinction	0.0044(5)	0.0040(4)	0.0085(4)
	coefficient
	Largest diff. peak	2.07/-1.90	2.54/-2.84	1.09/-0.79
	and hole/eA ˚-3

Table 3

 3 Atomic coordinates and equivalent isotropic displacement parameters/pm 2 of HP-Tb 2 Ni 2 Sn, TbNiSn, and HP-Dy 2 Ni 2 Sn U eq is defined as one-third of the trace of the orthogonalized U ij tensor with the tetragonal Mo 2 B 2 Fe structure. Similar experiments on Gd 2 Ni 2 Sn resulted in a new phase with a yet unknown structure. Most likely the gadolinium atoms are too large to enable formation of the tetragonal phase. Investigations on this new phase are in progress.In

	Atom	Wyck.	x	y	z	U eq/ U iso
	HP-Tb 2 Ni 2 Sn
	Tb	4h	0.17580(3)	x ? 1/2	1/2	74(1)
	Ni	4g	0.37915(9)	x ? 1/2	0	111(2)
	Sn	2a	0	0	0	83(2)
	TbNiSn
	Tb	4c	0.01175(4)	1/4	0.70253(4)	70(1)
	Ni	4c	0.3010(1)	1/4	0.4150(1)	96(2)
	Sn	4c	0.19108(6)	1/4	0.08665(6)	67(1)
	HP-Dy 2 Ni 2 Sn
	Dy	4h	0.17545(2)	x ? 1/2	1/2	154(1)
	Ni	4g	0.37918(7)	x ? 1/2	0	188(2)
	Sn	2a	0	0	0	162(1)

Table 4

 4 Interatomic distances/pm, calculated with the powder lattice parameters of HP-Tb 2 Ni 2 Sn and TbNiSn Within the three-dimensional [NiSn] network the Sn atoms have elongated tetrahedral SnNi 4/4 coordination, in contrast to the planar arrangement in HP-Tb 2 Ni 2 Sn. The Ni-Sn distances are all shorter than in HP-Tb 2 Ni 2 Sn, indicating stronger overall Ni-Sn bonding in the equiatomic phase.

	HP-Tb 2 Ni 2 Sn			TbNiSn
	Tb	2	Ni	280.9	Tb	1	Ni	302.2
		4	Ni	288.8		2	Ni	306.8
		4	Sn	327.9		2	Sn	311.2
		1	Tb	364.3		1	Sn	318.9
		2	Tb	371.7		2	Sn	320.5
		4	Tb	382.1		1	Sn	321.1
	Ni	1	Ni	250.4		2	Ni	328.3
		2	Tb	280.9		1	Ni	329.6
		4	Tb	288.8		2	Tb	364.8
		2	Sn	291.5		2	Tb	382.6
	Sn	4	Ni	291.5	Ni	2	Sn	259.0
		8	Tb	327.9		1	Sn	263.6
						1	Sn	278.9
						1	Tb	302.2
						2	Tb	306.8
						2	Tb	328.3
						1	Tb	329.6
					Sn	2	Ni	259.0
						1	Ni	263.6
						1	Ni	278.9
						2	Tb	311.2
						1	Tb	318.9
						2	Tb	320.5
						1	Tb	321.1

All distances within the first coordination spheres are listed. Standard deviations are all equal to or less than 0.3 pm structures as well

[12]

.

Acknowledgments This work was financially supported by the Deutsche Forschungsgemeinschaft.