Proc. Nati. Acad. Sci. USAVol. 87, pp. 4610-4614, June 1990Biochemistry

The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in a-like DNA polymerases is required by phage 429 DNApolymerase for protein-primed initiation and polymerization

(site-directed mutagenesis/metal binding site)

ANTONIO BERNAD, Jost M. LAZARO, MARGARITA SALAS*, AND LuIs BLANCOCentro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas, Universidad Aut6noma de Madrid, Canto Blanco, 28049 Madrid, Spain

Communicated by Severo Ochoa, March 12, 1990

ABSTRACT The a-like DNA polymerases from bacterio-phage 429 and other viruses, prokaryotes and eukaryotescontain an amino acid consensus sequence that has beenproposed to form part of the dNTP binding site. We have usedsite-directed mutants to study five of the six highly conservedconsecutive amino acids corresponding to the most conservedC-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our resultsindicate that in 429 DNA polymerase this consensus sequence,although irrelevant for the 3' -- 5' exonuclease activity, isessential for initiation and elongation. Based on these resultsand on its homology with known or putative metal-bindingamino acid sequences, we propose that in 029DNA polymerasethe Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of thedNTP binding site, involved in the synthetic activities of thepolymerase (i.e., initiation and polymerization), and that it isinvolved particularly in the metal binding associated with thedNTP site.

The amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser(YGDTDS) is found in a class of replicative DNA-dependentDNA polymerases known as a-like because of their sensi-tivity to aphidicolin (for review, see ref. 1) and severalnucleotide analogs (2-4) first thought to be specific inhibitorsofthe eukaryotic DNA polymerase a. To date, 17 a-like DNApolymerases, including the human DNA polymerase a (5-21), have been found to contain the YGDTDS motif, invari-ably located in the C-terminal portion. This motif and twoadditional consensus sequences, Val-Xaa-Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro and Asn-Ser-Xaa-Tyr-Gly, also present inthe C-terminal half of a-like DNA polymerases, have beenproposed to form the three-dimensional dNTP binding do-main (10, 11, 13, 14). In contrast, the N-terminal portioncontains three regions of lower amino acid similarity that arealso shared by DNA polymerases of other groups; it has beenproposed (22) that these regions, by structural and functionalhomology with the Escherichia coli DNA polymerase I (polI), form a conserved 3' -- 5' exonuclease active site.The high conservation of the YGDTDS motif in a-like

DNA polymerases from distantly related organisms suggeststhat the YGDTDS motif has a functional role; however, thelack of direct biochemical data has until now made thecatalytic significance of the YGDTDS motif rather specula-tive. A similar amino acid sequence motif, Tyr-(Gly/Met)-Asp-Asp, is present in otherDNA orRNA polymerasesthat are either DNA or RNA dependent (21). This homologyand the finding that amino acid substitutions within theTyr-Gly-Asp-Asp motif ofQB replicase reduced its activity invivo (23) led to the proposal ofa catalytic role ofthe YGDTDSmotif in polymerization (21). The fact that the YGDTDSsequence is not restricted to RNA- or DNA-primed DNA

polymerases but is also present in DNA polymerases forterminal protein-containing DNA genomes is of additionalinterest because it was not known whether the specificprotein-priming activity of these DNA polymerases had itsown catalytic site or shared general domains of the polymer-ization active site. Bacteriophage 429 DNA polymerase,product of viral gene 2 (Mr, 66,520), initiates replication(24-26) by catalyzing the formation of a phosphoester bondbetween the 8-OH group of Ser-232 in the terminal protein(p3) and 5'-dAMP, the 5' terminal nucleotide at both DNAends (27). The 429 DNA polymerase also has 5' -* 3'polymerization and 3'-* 5' exonuclease activities character-istic of other DNA replicases.Here we report results of site-directed mutagenesis in the

most conserved C-terminal motif(YGDTDS) and its effect onthe various enzymatic activities of the 429 DNA polymerase(for a preliminary report of some of these findings, see ref.28). Based on these results and the amino acid sequencesimilarity with other metal binding enzymes, we propose afunctional role for this motif in the 429 DNA polymerase andother a-like polymerases.

MATERIALS AND METHODSNucleotides, Proteins, and Templates. Unlabeled nucleo-

tides were purchased from Pharmacia P-L Biochemicals.[a-32P]dNTPs (410 Ci/mmol; 1 Ci = 37 GBq) were obtainedfrom Amersham. 429 terminal protein (p3) and protein p6were purified as described (29, 30). 429 DNA digested withrestriction endonucleases was prepared from proteinase K-treated 429 DNA (31). The 029 DNA-protein p3 complexand M13mpl9 single-stranded DNA were isolated as de-scribed (32, 33). The [32P]dA-tailed DNA was prepared byextending pUC19 (linearized with Pst I) with terminal deoxy-nucleotidyltransferase and [a-32P]dATP.

Site-Directed Mutants of 4029 DNA Polymerase. Among thepossible changes in the YGDTDS motif, we first selectedreplacements with minimal changes in secondary structure(proposed to form a ,-turn) according to the studies of Chouand Fasman (34) and Garnier et al. (35). We then selectedreplacements that would give conservative changes deducedfrom studies of a collection of crystallized proteins (36).Based on these criteria, five 429 DNA polymerase mutantscontaining the single changes [Tyr-454 -* Phe (Y454F),Cys-455 -> Gly (C455G), Asp-456 -* Gly (D456G), Thr-457Pro (T457P), and Asp-458 -* Gly (D458G)] were obtained andoverproduced (unpublished results). The wild-type and mu-tant 429 DNA polymerases were purified as described (22).

Activity Assays. The initiation reaction (i.e., protein p3-dAMP complex formation), DNA polymerase "minimal"

Abbreviations: YGDTDS, Tyr-Gly-Asp-Thr-Asp-Ser; pol I, DNApolymerase I.*To whom reprint requests should be addressed.

4610

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Proc. Natl. Acad. Sci. USA 87 (1990) 4611

assay (filling-in reaction), and 3' -- 5' exonuclease assayswere carried out as described (22). The replication assaysusing either 429 DNA-protein p3 or M13 DNA as templateswere carried out as described (37). The 429 terminal protein-429 DNA polymerase interaction assay using either wild-type or mutant 429 DNA polymerases was as described (38).Amino Acid Sequence Comparisons. The multiple alignment

of amino acid sequences (see Table 2) forming known orputative metal binding domains was obtained by a series ofbinary (pairwise) alignments, followed by shifting by eye toachieve maximal homology. The following conservativeamino acids were considered: isoleucine, leucine, and valine;aspartic acid, glutamine, and glutamic acid; lysine and argi-nine; serine and threonine; tyrosine and phenylalanine.

RESULTSInitiation Activity of 429 DNA Polymerase C-Terminal

Mutants. The 429 DNA polymerase mutants, overproducedand purified, were assayed by formation of the covalentcomplex between the terminal protein and 5'-dAMP (initia-tion reaction). As shown in Fig. 1A, whereas the change ofTyr-454 - Phe did not affect the activity, the changesCys-455 Gly and Asp-456 -* Gly gave 45% and 10%,respectively, of the wild-type value, and the changes Thr-457-> Pro and Asp-458 -* Gly essentially abolished the initiationactivity of the enzyme (see also Table 1 for quantitativevalues). As shown in Fig. 1B, the initiation activity of theC455G and D456G mutant polymerases was always lowerthan that of the wild-type enzyme over a wide concentrationrange of the activator metal ion (Mg2+). However, the profileobtained with the C455G mutant protein was clearly differentfrom that of the wild-type enzyme, probably indicating anincreased metal toxicity for the protein-priming reaction. TheT457P and D458G mutants were completely inactive over thesame concentration range of Mg2+ (data not shown). None ofthe mutations affected the interaction of the DNA polymer-ase with the terminal protein (data not shown), a steppreceding initiation. This fact, which is a criterion for con-servation of structure, strongly suggests that the loss ofactivity is at the level of the initiation reaction itself.

Elongation Activity of 429 DNA Polymerase C-TerminalMutants. The 029 DNA polymerase has been characterizedas a highly processive enzyme able to produce strand-displacement coupled to elongation (37). These particular

properties enable the wild-type 429 DNA polymerase toefficiently replicate the 429 DNA-protein p3 natural templategiving rise to full-length DNA (ref. 39 and Fig. 2A). More-over, the wild-type enzyme can replicate oligonucleotide-primed M13 DNA, using the circular template in a "rolling-circle" fashion, and produce DNA molecules greater thanunit-length M13 DNA (ref. 37 and Fig. 2B). In these tworeplication assays, only the C455G mutant was able to useboth DNA templates to generate elongation products similarin size to those obtained with the wild-type enzyme (Fig. 2and Table 1 for quantitative data). Considering that, forinitiation ofp3-DNA replication, this mutant is less efficient(45%) than the wild-type DNA polymerase, the value ob-tained in the 429 DNA-protein p3 replication assay (56%)seems to reflect the initiation efficiency and suggests anormal elongation activity. In agreement with this result, inthe absence of a protein-priming requirement, as in M13DNA replication, the elongation activities of the C455Gmutant and the wild-type enzyme were similar. As expected,the D456G, T457P, and D458G mutations that severelyaffected the protein-priming activity were unable to replicatethe p3-DNA template. However, the inability of these mu-tants to elongate the oligonucleotide-primed M13 DNAclearly indicates that Asp-456, Thr-457, and Asp-458 are alsoessential for the elongation activity of the 429 DNA poly-merase. The Y454F mutation does not affect the initiationactivity but gives rise to a polymerase that is unable toreplicate either a p3-DNA complex or primed M13 DNA.To analyze the effect ofthe various mutations on the ability

of the enzyme to catalyze a short polymerization reaction,EcoRI-digested 429 DNA was filled-in. This minimal poly-merization assay allowed us to study the ability of the 429DNA polymerase mutants to catalyze the incorporation offew nucleotides to the 3'-OH group of a DNA primer,diminishing other factors (processivity, ability to translocate,etc.) that could be affected, directly or indirectly, by thevarious mutations studied. As shown in Fig. 3 (see also Table1), the C455G mutant polymerase was even more active(290%) than the wild-type enzyme, and the Y454F and D456Gmutant proteins were clearly active (60% and 30%, respec-tively), whereas the T457P and D4586G mutant polymeraseswere inactive, even in this minimal polymerization assay.Similar results were obtained when the incorporation of othersingle deoxynucleotides was analyzed (not shown).

Bwt MUTANTSr---r

10 t

E 75-5

p3-dAMP-P-- -m -in-

YCOTD U- L L

Ln Ln Ln Ln-T

-IJL i C

LZD

a_

5SCL

2.5

Mg2,-[mM]1 2 5 10 20 40

_ _ ~~~Wt___ [4550

_ _ _ D4566

Mg2+ ,[mM]

FIG. 1. Effect of C-terminal point mutations on the protein-primed initiation activity of the 429 DNA polymerase. (A) The initiation assaywas carried out using 10 ng of wild-type or mutant 429 DNA polymerase in the presence of 10 mM MgCI2. After incubation for 2.5 min at 30'C,the p3-dAMP initiation complex formed was analyzed by SDS/polyacrylamide gel electrophoresis, autoradiographed, and quantitated (see Table1). (B) The initiation assay was carried out as described in A using 20 ng of wild-type or C455G or D456G mutant 429 DNA polymerase in thepresence of MgCl2. (Inset) p3-dAMP bands as a function of Mg2+ concentration. Analysis and quantitation were as described in A.

A

Biochemistry: Bernad et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

L.L 11v a ~ CL

-4 UI ID _-k>- 1W i I > L

p3-DNA- - * --4-

B

time 4 4 8 4 8 4 -

IL L CD CL aL', 141 -- co

Ln UI. LDn LD

*. 5t --t T

- h% ~ j

M13 DNA-a

time 5 15 15 5 1-.5 fly 815

FIG. 2. Effect of C-terminal point mutations on the elongationactivity of the 429 DNA polymerase. The replication assays withphage 429 DNA-protein p3 (A) or primed-M13 DNA (B) as templateswere carried out using 10 ng of wild-type (wt) or mutant 429 DNApolymerases as indicated. After incubation for the indicated times at30TC, the samples were processed and analyzed by alkaline agarosegel electrophoresis and autoradiography. The positions of unit lengthp3-DNA and M13 DNA are shown. A control without DNA poly-merase (lane -) was also carried out.

3' -+ 5' Exonuclease Activity of 429 DNA PolymeraseC-Terminal Mutants. To study the effect of point mutationsin the YGDTDS motif on the 3' -+ 5' exonuclease activity ofthe 429 DNA polymerase (40), a 3'-[32P]dA-tailed DNA wasused as substrate in the conditions described (22). As shownin Table 1, the five C-terminal mutants could digest thesingle-stranded 3' tail, their 3' -* 5' exonuclease activitybeing even higher than that of the wild-type enzyme. In allcases, the exonuclease activity detected was shown byspecific inhibition with anti-+29 DNA polymerase IgG (datanot shown) not to be due to other contaminant exonucleases.

Functional Significance of the YGDTDS Motif: HomologySearch. The YGDTDS motif (so called Asp-Asp motif) char-acterized by a Tyr-Gly-Asp-(Thr)-Asp core flanked by hy-

Table 1. Properties of mutant 429 DNA polymerasesReplication, %

Initiation, M13 Filling- 3' - 5'Enzyme % p3-DNA DNA in, % Exo, %

WT 100 100 100 100 100Y454F 107 8 <2 60 149C455G 45 56 110 290 255D456G 10 1.8 <2 30 130T457P 0.5 2.2 <1 1.3 131D458G <0.5 1.7 <1 <1 198

WT, wild type; Exo, exonuclease. The consensus sequence is(I/V)YGDTDS(I/V) and the 429 DNA polymerase sequence (resi-dues 453-460) is IYCDTDSI.

FIG. 3. Effect of C-terminal point mutations on the catalysis of asingle polymerization step by the 429 DNA polymerase. The DNApolymerase "minimal" assay with EcoRI-digested 429 DNA as

template was carried out using 10 ng of wild-type (wt) or mutant 429DNA polymerases as indicated. A control without DNA polymerase(lane -) was also carried out. After incubation for 1 min at 30'C, thesamples were processed and analyzed by native agarose gel electro-phoresis, ethidium bromide staining (Upper), and autoradiography(Lower). The position of the EcoRI fragments is shown.

drophobic spans has been proposed to be specific for manypolymerases (21). These conserved aspartates on an exposedloop in a predicted P3-hairpin structure have been proposed tobind magnesium as well as to act catalytically in the poly-merization process. Therefore, we compared this motif withamino acid sequences known to bind metal cations. Severalhelicases and NTP-utilizing enzymes, having associated NT-Pase activity, have a conserved amino acid segment close tothe "B" site of the NTP motif, probably involved in inter-action with the Mg2+ coordinated with the pyrophosphatemoiety of NTP (for review, see ref. 5). Table 2 shows thealignment of the corresponding segment of four bacterialhelicases and five putative RNA virus helicases, with theYGDTDS motif displayed by 17 known a-like DNA poly-merases (see also consensus 1) and with the consensus of theAsp-Asp motif (21) found in viral reverse transcriptases(consensus 2) and RNA replicases (consensus 3).Also shown in Table 2 is one of the amino acid segments

directly involved in metal binding at the 3' -+ 5' exonuclease

active site of E. coli pol I (7, 8), which is also conserved in theN terminus of a polymerase-like DNA polymerases (22).Interestingly, this conserved segment (named Exo I segment)has significant homology with the YGDTDS motif, probablyinvolved in the synthetic functions of a-like DNA polymer-ases. Furthermore, the residues directly involved in metalbinding are a conserved aspartate and glutamate correspond-ing to pol I amino acids 355 and 357, respectively (7, 8). X-raycrystallographic analysis has also shown the residues directlyinteracting with the dNTP substrate at the E. coli pol Ipolymerization active site; again, two residues, Gln-708 andGlu-710, are involved in binding a magnesium ion (L. Beese,J. Friedman, and T. A. Steitz, personal communication).There are other enzymes ofknown tertiary structure where an

aspartic or glutamic acid (or glutamine) or both bind a metal iondirectly, as is the case with phosphoglycerate kinase (ref. 9;see Table 1), concanavalin A (41), E. coli elongation factorEF-Tu (42), and p21, the product of the ras gene (43).

In summary, the homology detected strongly suggests thatthe YGDTDS motif plays a role in metal binding at thepolymerase active site of a-like DNA polymerases and, inparticular, the two aspartic acid residues; however, variantsof this motif (in which the Asp-Asp pair can be substituted by

A -4 ;.: " -A. .- XD

j-4 L--

^ >- _ O r

~A--B-"

D-

II-

S6

4612 Biochemistry: Bernad et al.

Proc. Natl. Acad. Sci. USA 87 (1990) 4613

Table 2. Alignment of known and putative metal-binding motifsAmino acid

Metal-binding protein Ref. Position sequence

Helicase (bacterial)E. coli RecB 5, 6 407 TALLLIGDPKQA1YAE. coli UvrD 5, 6 241 GU1MIVMDDQSIXSE. coli RecD 5, 6 290 ABFYELGDRQLASVE. coli Rep 5, 6 235 AEFTVVaDDQSIYS

Putative helicase(RNA virus)BNYVV p237 5,6 1027 TIYLV-GDE.QQGIQCMV 5,6 803 RALCF-GDSEQIAESBMV 5, 6 776 QVLAF-I-GEQIAFSAIMV 5, 6 930 EVIGE-GDTEQIPEYTMV 5,6 927 IAYYGD.TQQIPYI

DNA polymerase I(E. coli) (Exo I) 7, 8 348 KAPYFAFD.TERLDN

Yeast PGK 9 365 TVIGGGDAITYAKa-like DNA polymeraseT4 10 612 DFIAA-GDTD-SVYV029* 11 450 DRIIY-CDTD-SIHLPRD1* 10 422 EMPLY-CDTD-SIICS1* 10 711 DDCYY-TDTD-SVV.pGKL1* 10 868 AECIYSDTD-SIFVpAI2* 12 1037 ADNLYAVDTD-GIKYAdenovirus 2* 13 864 LKSVY-GDTD-SLFVVaccinia 14 720 FBSVY-GDTD-SVFTFowl-pox 15 729 FBSVY-GDTD-SIFSVZV 10 845 VKVIY-GDTD-SVFIHSV-1 13 880 MRIIY-GDTD-SIFVEBV 13 749 LRVIY-GDTD-SLFIHCMV 16 904 ERVIY-GDTD-SVFVAcMNPV 17 662 FKVVY-GDTD-STFVYeast CDC2 18 752 DAVVY-GDTD-SYMYYeast pol l 19 990 LLVVY-GDTD-SVMIPolymerase a (human) 20 996 LEVIY-GDTD-SIMIConsensus 1 nnhhY GDTD Shhh

Reverse transcriptaseConsensus 2 21 nhhnY MD D hhhn

RNA replicaseConsensus 3 21 nhhnY GD D nhhn

Alignment of known or putative metal binding amino acid motifsfrom NTP- and dNTP-utilizing enzymes. The position number indi-cates the first residue in each sequence relative to the N-terminal endof each polypeptide. In most cases the references given indicatewhere the sequence or homology was first reported. Residuesconserved in most of the sequences compared are underlined. Insome cases consecutive residues are separated by hyphens. Simi-larities between the bacterial helicases and putative helicases fromRNA virus were noted by the original authors. Consensus 2 and 3,corresponding to viral reverse transcriptases and RNA replicases,respectively, were according to Argos (21). In the consensus, hmeans hydrophobic and n means any amino acid residue. BNYVV,beet necrotic yellow vein virus; CMV, cucumber mosaic virus;BMV, brome mosaic virus; AIMV, alfalfa mosaic virus; TMV,tobacco mosiac virus; PGK, phosphoglycerate kinase; VZV, vari-cella zoster virus; HSV-1, herpes simplex virus 1; EBV, Epstein-Barr virus; HCMV, human cytomegalovirus; AcMNPV, Au-tographa californica mononuclear polyhedrosis virus. The single-letter amino acid code is used.*Terminal protein-containing genome.

other residues capable of contributing carboxyl oxygens asligands) seem to play a similar role at the catalytic site of avast class of NTP and dNTP-utilizing enzymes.

DISCUSSIONThe 429 DNA polymerase, which consists of a single poly-peptide of Mr 66,520, is a member of a group of enzymesnamed a-like DNA polymerases that includes DNA-dependent DNA replicases from prokaryotes to higher eu-

karyotes. Comparison of the amino acid sequences of thesefunctionally related proteins revealed several segments in theC-terminal portion with a remarkable degree of amino acidsequence similarity. We now report functional data on thecatalytic significance of five consecutive amino acids in theYGDTDS motif corresponding to the most conserved C-terminal segment present in a-like DNA polymerases. Site-directed point mutants in the YCDTD sequence of the 429DNA polymerase essentially did not affect the 3' -- 5'exonuclease activity of the enzyme. In agreement with thisobservation, the 3' -+ 5' exonuclease active site of the 429DNA polymerase and many other prokaryotic and eukaryoticDNA polymerases is located in the N-terminal portion form-ing a separate domain with structural and functional homol-ogy to the corresponding active site of E. coli pol I (22). Onthe other hand, the processive elongation activity of the 429DNA polymerase was completely inhibited by all mutationswith the exception of C455G, which gives the 4)29 DNApolymerase the complete consensus in the YGDTDS motif.However, when the effect of the different mutations onnonprocessive elongation was determined, the Y454F,C455G, and D456G mutant proteins were active and only theT457P and D458G mutations appeared to be critical for aminimal DNA polymerase activity.The initiation activity ofthe 429DNA polymerase was also

essentially abolished in mutants T457P and D458G and se-verely affected by the D456G mutation, although none ofthese mutations affected the ability of the enzyme to interactwith the terminal protein. The results obtained with mutantY454F could be explained if the mutation affects the propertranslocation of the 4)29 DNA polymerase, having less effecton "one-step" catalytic processes, such as initiation orfilling-in aDNA end, than in processes involving the couplingof consecutive catalytic events such as 4)29 or M13 DNAreplication; similar results were obtained when Tyr-454 wasreplaced by Ser (unpublished results). It is interesting to notethat mutation D456G affects the protein-priming reaction toa greater extent than the filling-in reaction relative to theY454F mutation, indicating that, although the active site forinitiation and polymerization seems to be shared, specificdifferences may exist. In agreement with this, the glycine inthe YGDTDS motif is not a completely conserved residue;variants in this position are always present in DNA poly-merases from terminal protein-containing genomes (see Ta-ble 2). This fact can be related with the reduced initiation butnormal elongation activity of the C455G 429 DNA polymer-ase mutant. Interestingly, this mutant showed differences inthe maximum of activating metal ion concentration withrespect to the wild-type protein; again, this effect was specificfor the catalysis of the protein-priming reaction.

All these results and the sequence similarity observedbetween the YGDTDS motif and several known or putativemetal-binding motifs led us to conclude that the YGDTDSmotif plays a catalytic role in the processes of synthesis(initiation and/or elongation) of a-like DNA polymerases,probably being involved in metal binding at the dNTP site.For the 429 DNA polymerase and in agreement with thehypothesis proposed by Argos (21), the second aspartate(Asp-458) was critical (probably being involved in catalysisand/or binding magnesium), whereas the first aspartate (Asp-456) was not an absolute requirement for catalysis. In addi-tion, Thr-457, highly specific in the consensus motif corre-sponding to the a-like DNA polymerases, was also essentialfor both synthetic activities of the 429 DNA polymerase.The presence of the YGDTDS motif and several other

conserved segments (10, 20, 22) in both protein-primed DNApolymerases and other DNA polymerases suggests that theyare derived from a common ancestral gene. Thus, a plausiblehypothesis is that the protein-priming function was acquiredby modifications of the general polymerization domain pres-

Biochemistry: Bernad et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

ent in an ancestral DNA polymerase. The results presentedsupport this hypothesis and the existence in protein-primedDNA polymerases of a common active site for initiation andelongation activities.

This investigation has been aided by Research Grant 5 R01GM27242-10 from the National Institutes of Health, by Grant PB87-0323 from Direcci6n General de Investigaci6n Cientifica y Tdcnica,and by a grant from Fundaci6n Ram6n Areces. A.B. was a Post-doctoral Fellow of the Spanish Research Council.

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