Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes

    Dual function of Ixr1 in transcriptional regulation and recognition of cisplatinDNA adducts is caused by differential binding through its two HMG-boxes A. Vizoso-V´azquez, M. Lamas-Maceiras, R. Fern´andez-Leiro, A. RicoD´ıaz, M. Becerra, M.E. Cerd´an PII: DOI: Reference:

S1874-9399(16)30254-1 doi:10.1016/j.bbagrm.2016.11.005 BBAGRM 1105

To appear in:

BBA - Gene Regulatory Mechanisms

Received date: Revised date: Accepted date:

1 July 2016 16 November 2016 17 November 2016

Please cite this article as: A. Vizoso-V´azquez, M. Lamas-Maceiras, R. Fern´andezLeiro, A. Rico-D´ıaz, M. Becerra, M.E. Cerd´an, Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes, BBA - Gene Regulatory Mechanisms (2016), doi:10.1016/j.bbagrm.2016.11.005

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ACCEPTED MANUSCRIPT Dual function of Ixr1 in transcriptional regulation and recognition of cisplatinDNA adducts is caused by differential binding through its two HMG-boxes

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Vizoso-Vázquez A.1, Lamas-Maceiras, M.1, Fernández-Leiro R.2, Rico-Díaz A.1,

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Becerra M. 1 and Cerdán M.E.1&

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1 Universidade da Coruña, Grupo EXPRELA, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de Bioloxía Celular e Molecular, Facultade de

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MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

Corresponding author

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&

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Ciencias, , A Coruña, Spain

Laboratorio de Bioquímica y Biología Molecular

Universidad de A Coruña 15071 A Coruña (SPAIN)

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Facultad de Ciencias

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e.mail: [email protected]

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Phone: 34 881 012141

ACCEPTED MANUSCRIPT ABSTRACT Ixr1 is a transcriptional factor involved in the response to hypoxia, which is also related to DNA repair. It binds to DNA through its two in-tandem high mobility group

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box (HMG-box) domains. Each function depends on recognition of different DNA structures, B-form DNA at specific consensus sequences for transcriptional

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regulation, or distorted DNA, like cisplatin-DNA adducts, for DNA repair. However, the contribution of the HMG-box domains in the Ixr1 protein to the formation of different protein-DNA complexes is poorly understood. We have biophysically and biochemically characterized these interactions with specific DNA sequences from the promoters

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regulated by Ixr1, or with cisplatin-DNA adducts. Both HMG-boxes are necessary for transcriptional regulation, and they are not functionally interchangeable. The in-tandem arrangement of their HMG-boxes is necessary for functional folding and causes

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sequential cooperative binding to specific DNA sequences, with HMG-box A showing a higher contribution to DNA binding and bending than the HMG-box B. Binding of Ixr1 HMG boxes to specific DNA sequences is entropy driven, whereas binding to

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platinated DNA is enthalpy driven for HMG-box A and entropy driven for HMG-box B. This is the first proof that HMG-box binding to different DNA structures is associated

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with predictable thermodynamic differences. Based on our study, we present a model to explain the dual function of Ixr1 in the regulation of gene expression and recognition

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of distorted DNA structures caused by cisplatin treatment.

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Keywords: thermodynamics; protein-DNA interactions, SOX transcriptional factors, DNA repair, HMGB proteins, Saccharomyces cerevisiae

ACCEPTED MANUSCRIPT 1. Introduction Proteins influence cellular functions through specific interactions with other proteins and biomolecules. These interactions that govern the formation of different

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complexes are also important in understanding their multiple roles in many processes [1, 2]. High mobility group B (HMGB) proteins are non-histone proteins that associate

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with chromatin and contain a versatile eukaryotic DNA binding domain - the HMG-box [3, 4]. Based on structural and phylogenetic studies, 2 broad subfamilies of HMGB proteins have been defined; the first includes those that bind to DNA with low or without sequence specificity (NSS) and have, with some exceptions, 2 or more in-

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tandem HMG-box domains. Their role is related to chromatin modification, participating in many nuclear functions, such as co-activation or silencing of transcription and V(D)J junction recombination [5, 6]. The second includes proteins

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that bind to DNA by recognizing a specific DNA sequence (SS). They usually contain a single HMG-box domain and are transcription factors expressed only in a few cell types. Despite these differences, both subfamilies of HMG-box proteins can bind to

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B-form DNA through the minor groove, thereby inducing wide DNA bending [5, 6]. Ixr1 is one among the 7 HMGB proteins of Saccharomyces cerevisiae. ROX1

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and IXR1 are considered SOX genes, encoding transcription factors, with one and 2 HMG boxes in-tandem, respectively [7]. SOX stands for SRY-related HMG box, and Sry is a gene involved in human sex determination, which resides in the Y-

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chromosome [7]. Ixr1 has been characterized as a transcriptional regulator of yeast responsiveness to changes in oxygenation [8, 9]. It is also necessary for maintaining

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ribonucleotide reductase (RNR1) expression, and consequently dNTP pools for DNA synthesis in both the normal cell cycle and after DNA damage [ 1 0 ] . Other HMGB yeast proteins that recognize and bind cisplatin adducts, like Nhp6A and Nhp6B, facilitate the repair of these lesions [11]. Yeast cells devoid of these both proteins are indeed more sensitive to cisplatin, a drug that preferentially forms 1-2 intra-strands adducts with DNA [11]. Inversely, ixr1∆ null mutant has greater resistance to cisplatin, which raises the hypothesis of whether Ixr1 can block repair of the major cisplatinDNA adducts in vivo [12]. An alternative, but compatible, model to explain increased resistance to cisplatin in the ixr1∆ null mutant has been suggested, in which elimination of Ixr1 creates endogenous stress, pre-activating the genome integrity checkpoint above basal levels. Cells with a constitutively activated checkpoint more efficiently repair DNA lesions such as those caused by cisplatin [13]. Ixr1 binding to platinated DNA was described over 20 years ago [14]. More recently, recognition of specific D N A sequences in the promoters of the regulated

ACCEPTED MANUSCRIPT genes HEM13, ROX1 and TIR1 by Ixr1 has been proved both in vitro and in vivo [15, 16]. It is known that the existence of 2 HMG-box domains in- tandem is crucial in determining the specific functions of other HMGB proteins, e.g. human HMGB1 [17] and TFAM [18], or the yeast protein Hmo1 [19]. However, biophysical and

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biochemical characterization of the interactions between each HMG-box domain present in Ixr1 and its recognized DNA targets (specific regulatory sequences in the

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promoters or cisplatin-DNA adducts) have not been explored. This is needed to understand the different molecular mechanisms by which this protein controls DNA repair and transcription, and to learn how each HMG-box domain contributes in its own way to the recognition of its different DNA targets.

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In trying to improve our understanding of the significance of the 2 in-tandem HMG-box domains in the dual function of Ixr1 in transcription and DNA repair, we

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have compared single and double domain interactions with many types of DNA sequences/structures by biochemical and biophysical experimental approaches. The interdependence of the 2 Ixr1 HMG-box domains needed to get functional domain

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folding was also tested in the presence or absence of DNA.

2.1. Nomenclature

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2. Materials and methods

Genes from S. cerevisiae are indicated in capital letters and italics. Proteins

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from S. cerevisiae are indicated in lower case (except for capital letters). Proteins from other organisms are indicated in capital letters, following international nomenclature

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guidelines.

2.2. Oligonucleotides and Plasmids The sequences of oligonucleotides purchased from Isogen Lifes Sciences, Inc

that we used are summarized in supplemental Table S1. The constructs Ycplac33IXR1 (full-length Ixr1 ORF plus 1000 bp upstream and downstream) and pKLSL150tandem-AB (residues 338-510) were produced by PCR using primers ECV775AV and ECV776AV for Ycplac33-IXR, and ECV683hmgf and ECV785AV for pKLSL150tandem-AB, and ligation into Ycplac33 (between the BamHI and HindIII restriction sites) [20], and pKLSL150 plasmid (SacI and XhoI restriction sites) [21], respectively. The constructs Ixr1_HMGAΔ, Ixr1_HMGBΔ and Ixr1_HMGA/BΔ were obtained by divergent PCR, using the primer pairs ECV779AV + ECV780AV, ECV781AV + ECV782AV and ECV780AV + ECV781AV, respectively, and with the Ycplac33-IXR1 as template. The constructs Ixr1_HMGAx2 and Ixr1_HMGBx2 were produced by homologue recombination using the primer pairs AVV101 + AVV102

ACCEPTED MANUSCRIPT and AVV99 + AVV100, respectively. Finally, the constructs encoding individual HMGbox domains A (residues 338-439) and B (residues 430-510) were produced by the one-step PCR-based method of Qi et al. [22], using the primers HMGAF and HMGAR for Ixr1_HMGAΔ construction, and HMGBF and HMGBR for Ixr1_HMGBΔ

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construction (see Table S1) and with pKLSL150-tandemAB as template. PCR reactions involved Vent polymerase (New England Biolabs). All constructions were

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verified by DNA sequencing.

2.3. Expression and purification of HMG-box A, HMG-box B and HMG-box tandem A/B

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The proteins were overexpressed in BL-21(DE3) cells (Novagen) and 2xTY medium by induction with 1 mg/mL IPTG for 3 h at 37oC and 200 rpm shaking. Cell

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pellets were collected and lysed by sonication in high salt lysis buffer [50 mM sodium phosphate (pH 6.9), 1 M NaCl, 2 mM DTT, 2X complete protease inhibitor cocktail (Roche)]. After centrifugation, supernatants were passed through Sepharose CL-6b ®

column (GE Healthcare) pre-

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resin (Sigma-Aldrich) packed into a Tricorn

equilibrated in buffer A [50 mM sodium phosphate (pH 6.9), 200 mM NaCl, 2 mM DTT, 1 mM EDTA]. Proteins were eluted by linear gradient of lactose (0-300 mM)

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in buffer A and loaded on a HisTrap HP 5 mL column (GE Healthcare) preequilibrated in wash buffer B [50 mM sodium phosphate (pH 6.9), 200 mM NaCl, 2

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mM DTT] for a second elution by linear gradient of imidazole (0-500 mM) in buffer B. Polypeptides were dialyzed with buffer C [50 mM sodium phosphate (pH 6.9), 100

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mM NaCl, 2 mM DTT, 1 mM EDTA], and simultaneously digested with TEV protease (Sigma-Aldrich) overnight at 8ºC. After digestion, proteins were further purified by gel filtration chromatography using a Hi-load Superdex 200 16/60 column (GE Healthcare) pre-equilibrated with buffer C. Proteins were concentrated by ultrafiltration using Amicon® Ultra-15 Centrifugal Filters, 3 kDa (Merk-Millipore). Sequences of the purified regions of Ixr1 are shown in supplemental material Fig. S1A, and the homogeneity and integrity of purified proteins were examined by SDSPAGE (15% w/v polyacrylamide gel) electrophoresis (Fig. S1B). The secondary structural content of purified proteins was checked by Far-UV CD, confirming the anticipated profiles (Fig. S2). A lower content in α-helix in the HMG-box A construction was attributable to the amino terminal tail included during the design, since it was necessary for protein stabilization, corresponding to the unfolded region between boxes A and B.

ACCEPTED MANUSCRIPT 2.4.qPCR Total RNA isolated with the GeneJET RNA Purification Kit (Thermo Scientific) was converted into cDNA and labelled with KAPA SYBR FAST universal one-step qRT-PCR kit (Kapa Biosystems, Inc, Woburn, Massachusetts, USA). Primers were

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designed to generate 60-85 base-pair amplicons, with a Tm of 59 or 60°C. ECO RealTime PCR System was used for the experiments and the calculations (Illumina, Inc.,

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San Diego, California, USA). Two independent RNA extractions and 2 technical replicates were assayed for each strain or condition. The mRNA levels of selected genes were corrected by the mRNA levels of TAF10, a gene previously verified to be constitutive under these conditions, and unaffected by ixr1∆ deletion. Relative

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expression was calculated using the Pfaffl method [23]. Statistical significance of the

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results involved student’s t-test for independent samples.

2.5. Western-blot analysis

The different constructs in section 2.2 were tagged with FLAG epitope by

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divergent PCR, using the primer pairs AVV241 + AVV242 (Table S1). Aliquots of W303 ixr1∆ cell cultures containing the different constructs of Ixr1 were taken at OD600

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= 0.8, collected by centrifugation and resuspended in buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 2.5 mM EDTA, 1 % (v/v) Triton X-100, 1 mM PMSF and 1 x Complete

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Protease Inhibitor (Roche)]. Cells lysed using glass beads (Sigma-Aldrich) were quickly spun to sediment debris. Protein concentration (Bradford reagent, Bio-Rad) of the lysates were adjusted and loaded onto a 10% (w/v) polyacrylamide gel, followed by

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blotting on PROTRAN® nitrocellulose membranes (Whatman®). Anti-FLAG mouse IgG antibody (Sigma-Aldrich) and goat anti-mouse IgG-HRP (scI2005; Santa Cruz Biotechnology) primary and secondary antibodies, respectively, were used. BM Chemiluminiscence Western Blotting kit IMouse/Rabbit (Roche) was used to detect the different Ixr1 protein versions in Amersham HyperfilmTM ECL High Performance Chemiluminiscence Films (GE Healthcare). Membrane washes, incubation times and developing procedures followed the manufacturer’s instructions (BM Chemiluminiscence Western Blotting kit IMouse/Rabbit (Roche). Band quantification and analysis used ImageLab software from Bio-Rad (version 5.3).

2.6. Electro Mobility Shift Assay (EMSA) DNA duplexes were prepared by mixing the complementary oligonucleotides in equimolar amounts, heating to 95oC for 5 min and cooling slowly to room temperature in darkness. Solutions of DNA for the experiments were prepared by

ACCEPTED MANUSCRIPT extensive dialysis against the solvent, as necessary. DNA platination was by the protocol of Cohen et al. [24]. Reaction mixtures (15µl) containing 10 nM DNA 5’ labelled with FAM were made in 20 mM potassium phosphate buffer, pH 6.8, 100 mM KCl, 1 mM EDTA, 2 mM DTT, 5% (v/v) glycerol, 500 µg/ml bovine serum

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albumin, and with protein at the different concentrations indicated. Samples were electrophoresed as previously described [16], and the gels scanned for fluorescence

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in a Typhoon® FLA 7000 biomolecular imager v.1.2 (GE Healthcare) to detect the FAM fluorophore, using 473 nm laser excitation and a Y520 filter.

2.7. Fluorescence anisotropy (FA)

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Fluorescein-labelled (FAM) dsDNA oligonucleotides (Table S1: AVV190AVV191, AVV212+AVV213, AVV214-AVV215 and AVV218-AVV219) were

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extensively dialyzed (10 mM K2HPO4 pH 6.8, 100 mM KCl, 2 mM DTT, 1 mM EDTA, 500 µg/ml bovine serum albumin). Fluorescence anisotropy titrations were performed at 25oC on a Multi-modal SynergyTM H1 plate reader (Biotek), using

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384-well NBS™ Microplate (Corning) with 15µl per well. The excitation and detection wavelengths were 485 and 528 nm, respectively, with a dichroic mirror (510 nm) and polarizer filter assembly. Tumbling rates or changes in the rotational times of the

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small labelled-DNAs when tightly bound to large proteins were used to calculate fluorescence anisotropy values. In each titration, the fluorescence anisotropy of a

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solution of 50 nM fluorescein-tagged duplex DNA was measured and represented as a percentage of ligand bound as a function of the added protein concentration. For

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each competition experiment, the polarization signal was followed in time, which indicated that a 30 min incubation period was adequate to reach equilibrium (data not shown). Binding data were fitted by non-linear least-squares regression, using GraphPad Prism 6.0 (GraphPad software). Each titration was run 3 times, and the final Ka was taken as the mean. Gibbs energies of association, as also their splitting into non-electrostatic and electrostatic components, were calculated as before [25].

2.8. Isothermal calorimetry (ITC) ITC experiments used a MicroCal ITC-200 machine (GE Healthcare). Protein and DNA samples were extensively dialyzed against 3 L buffer (10 mM K2HPO4, pH 6.8, 100 mM KCl, 0.5 mM TCEP) and degassed before each measurement. Different DNA samples at suitable concentrations (100-500 µM) were titrated in 2.5 µL injections to the cell containing the protein at concentrations from 20 to 40µM. The raw heats of injection were measured as the cell was stirred at 1000 rpm at 25oC. DNA was titrated into buffer as a control, and the heat evolved was subtracted

ACCEPTED MANUSCRIPT from the heat for the protein/DNA injections. The heat of injection measured from the first titration point was discarded. Raw heats of injection were baseline- corrected, integrated with respect to time, and fit to a binding isotherm to calculate the dissociation constants and energies of enthalpy of the reaction, using Origin® v7 SR4

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scientific plotting software (OriginLab Corporation). Data were processed and

and explained in the supplemental material.

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adjusted to the binding model given in Figure S6 as previously described [26, 27],

2.9. Förster resonance energy transfer (FRET) and intrinsic tryptophan fluorescence Experiments were conducted with a DNAROX1 duplex template that was 3 -

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labelled in one strand with TAMRA (AVV228, acceptor), and with FAM (AVV190, donor) in the other after they had been extensively dialyzed (10 mM K2HPO4 pH 6.8, 100 mM KCl, 2 mM DTT, 1 mM EDTA, 500 µg/ml bovine serum albumin).

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Fluorescence anisotropy titrations were performed at 25 oC on a Multi-modal Synergy® H1 plate reader (Biotek) using 384-well Well Low Volume Black Round

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Bottom Polystyrene NBS™ Microplate (Corning) with 15 µl per well. FRET effect (FE) and bending angles were calculated as before [28]. The values of εFAM490/εTAMRA560 and εTAMRA490/εTAMRA560 were calculated to be 0.149 and

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0.071 for DNAROX1 duplex, respectively. The Dr (Förster radius) value calculated, as previously described [25], for DNAROX1 was 49.8 Å. In the absence of protein,

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the calculated Doda value for DNAROX1 was 75 Å. Intrinsic fluorescence measurements were recorded using an excitation

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wavelength of 280 nm and emission wavelength ranging from 300 to 400 nm. Slit widths for both excitation and emission were kept at 5 nm. Concentrations of individual HMG-box domains and DNAROX1 were 1 µM in buffer containing 10 mM K2HPO4 pH 6.8, 100 mM KCl, 2 mM DTT, and 1 mM EDTA, 500 µg/ml bovine serum albumin.

2.10. NMR spectroscopy Several changes were introduced in the purification protocol to obtain the 15Nlabelled proteins for the NMR experiments. Bacterial cultures of BL21(DE3) were grown in MOPS minimal medium prepared as before [29]. For the preparation of

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labelled protein, 100 mM 15NH4Cl was used as the sole nitrogen source, as appropriate. After digestion with TEV protease, proteins were purified using a Heparin HP 5 mL column (GE Healthcare) pre-equilibrated with wash buffer D [20 mM sodium phosphate (pH 6.8), 50 mM KCl, 2 mM DTT, 1 mM EDTA] by linear gradient

ACCEPTED MANUSCRIPT of NaCl (0-500 mM)]. Proteins were extensively dialyzed in buffer E [10 mM sodium phosphate (pH 6.5), 50 mM KCl, 2 mM DTT, 1 mM EDTA] before concentration by ultrafiltration, using Amicon® Ultra-15 Centrifugal Filters, 3 kDa (Merk-Millipore). NMR measurements were made on samples containing 0.5 mM 15

2

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N-labelled protein or

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N-labelled protein-DNA complex, 10% H2O in 10 mM sodium phosphate (pH 6.8),

0.1 mM EDTA and 1 mM TCEP (Tris (2- carboxyethyl) phosphine hydrochloride)

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(Sigma-Aldrich). 1D 1H and 1H-15N HSQC spectra experiments were carried out at 298 K on a Bruker Avance III AV600 spectrometer equipped with a quad-resonance HCNF probe head and actively shielded z-gradients. Data were processed with the AZARA suite of programs (v. 2.8, 1993-2013; Wayne Boucher and Department of

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Biochemistry, University of Cambridge, unpublished).

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2.11. Circular dichroism (CD) spectropolarimetry and thermal melting Circular dichroism measurements involved a JASCO spectropolarimeter (J815) with a thermostatically controlled cell holder attached to a Peltier PTC-423S system. All proteins were extensively dialyzed (10 mM K2HPO4 pH 6.8, 50 mM NaF,

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2 mM DTT, 1 mM EDTA). Spectra were collected at 5oC in a continuous scanning mode, with a scanning rate of 50 nm/s and a response time of 2 s. Each spectrum

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was the average of 10 scans. Far-UV CD spectra were taken in the range of 190-260 nm with 1 nm steps in a cell of 0.1 cm path length. From the raw data, the collected

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spectra were buffer-subtracted and converted from millidegrees (Θobs, mdeg) to -1 -1 molar ellipticity ([Θ], degrees cm2 dmol residue using the equation:

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[Θ] = (Θobs x M) / (10 x l x C)

where M is the protein mean residue molecular weight, l is the optical path length of the cuvette in centimetres, and C is the concentration of the protein in mg mL-1. The percent secondary structure content for alpha class proteins was calculated by the K2D2 method [30] for α-helix class proteins. The temperature dependence of the circular dichroism spectra of the different proteins were determined upon continuous heating, with a rate of 1 K min-1 in the temperature range 5-95o C at 222 nm wavelength using a 0.1 cm path length sealed cell. Ellipticity signals were plotted as a function of temperature; non-linear Boltzmann fit was performed, and the melting temperature calculated as the maximum of the first derivative of this curve, using GraphPad Prism 6.0.

2.12. Differential Scanning Fluorometry Differential Scanning Fluorometry (DSF) was used to analyze the thermal

ACCEPTED MANUSCRIPT stability of the purified proteins, and compare the results of melting temperature with the data obtained by circular dichroism [31, 32]. A pre-screening of dye (Sypro Orange, Sigma-Aldrich) and protein was used to find the best dye and protein concentration, which was 20x Sypro Orange for 50 μM HMG-box proteins. 96-well thin-wall PCR

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plates (Thermo Scientific) sealed with Optical-Quality Sealing Tapes (Bio-Rad) were used. Samples were incubated for 5 min at 25ºC and then heated to 95ºC in

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increments of 0.5ºC x min-1 using an iCycler-Q real-time PCR machine (Bio-Rad). Fluorescence of the dye was monitored simultaneously using λex: 490 nm and λem: 530 nm filtered wavelengths. Fluorescence intensities were plotted as a function of temperature and a non-linear Boltzmann fit was applied with GraphPad Prism 6.0.

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Melting temperature was calculated as the maximum of the first derivative of this curve

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[31].

2.13. Bioinformatics analysis and 3-D modelling Alignment was done by the tool ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) using the PAM matrix and edited by

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ESPript 3 (http://espript.ibcp.fr/ESPript/ESPript/index.php). 3-D Homology modelling of Ixr1 HMG-box domains was done by Phyre2 server [33]

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(http://www.sbg.bio.ic.ac.uk/phyre2/html/ page.cgi?id=index) based on protein templates human HMGB1 (PDB 2YRQ), Tox2 protein from Mus musculus (PDB

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2CO9) and human HMGB1 (PDB 2E6O) in the Protein Data Bank (http://www.rcsb.org). Protein model pictures were made with the PyMol package

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(v1.7) (www.pymol.org).

3. Results

3.1. Characteristics of the HMG-box domains present in Ixr1 In Ixr1 protein, the 2 HMG boxes are organized in tandem and extend from positions 360 to 430 (HMG-box A) and 433 to 503 (HMG-box B). They share just 24.6% identity, which may lead to functional differences between them. An earlier paper [16] reported that Ixr1 HMG-box A aligns better with the first HMG domain of rat HMGB1, an abundant protein that binds DNA without sequence specificity (UniProt ID: P63159). However, the best alignment of HMG-box B was with the single HMG-box domain of the transcriptional factors Rox1 and SRY, from yeast and human, respectively, which bind specific DNA sequences (SS). Modelling of the 2 Ixr1 HMG-box domains shows the characteristic L-shaped fold formed by 3 α-helices with an angle of ~80o between the 2 arms. The long arm or minor wing is composed of the extended N-terminal strand and third α-helix, whereas first and second α-

ACCEPTED MANUSCRIPT helixes form the short arm or major wing (Fig.1B) A multiple alignment of only proteins containing 2 HMG-box domains in tandem (the S. cerevisiae paralog ARS-binding factor 2 (Abf2), the dorsal repressor DSP1 from Drosophila melanogaster and 5 human proteins (such as HMGB1-4 and

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TFAM) shows that Ixr1 is more closely related to its paralog, Abf2, and to the human protein, TFAM (Fig.1A). Interestingly, Ixr1 and TFAM have been the only

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proteins that have both sequence-specific and non-sequence-specific DNA binding properties until now [14, 34, 35]. The other proteins in the alignment, as the majority of the proteins with 2 HMG-box domains, recognize DNA without sequence specificity [3, 36, 37]. Extension of the linker connecting the 2 in tandem HMG-box domains

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might condition their relative spatial position, and is therefore important for its function. Ixr1 and Abf2 present the shortest linker region in the alignment, with only 5

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amino acids (Fig. 1A, grey box).

Several HMGB proteins, Ixr1 among them, bind with high affinity to cisplatinmodified DNA [11, 12, 14, 38-41]. The phenyl ring of the F37 residue in human HMGB1 (UniProt ID: P09429) is located in the first HMG-box at the N- terminus of the

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second α-helix in the major wing, and it intercalates into the 1,2- intra-strand DNA adduct formed by cisplatin. F37 acts as a wedge by stacking onto the solvent-

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exposed face of the cisplatin-DNA adduct. Consequently, a HMGB1 F37A mutant is severely impaired from binding cisplatin-modified DNA [42]. This residue is

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conserved in the other human HMGB2-4 proteins (Fig. 1A, shaded in yellow) and in others not considered in this alignment, such as F48 in yeast Nhp6A [11]. However, this important phenylalanine is not conserved in any of the 2 HMG-box domains of

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Ixr1, Abf2 or TFAM (Fig. 1A). Instead, Ixr1 presents hydrophobic residues with aliphatic side chains (V388 and L461, respectively) in the equivalent positions of HMG-box domains A and B. Hydrophobic residues are usually involved in DNA intercalation, as in other human HMGB proteins such as HMGB1 [39, 43, 44], SSRP1 [44], TFAM [44] and UBF [45], but their role in interacting with cisplatin-modified DNA has not been reported. In summary, although Ixr1 acts as a transcriptional factor and recognizes SS targets, it has 2 HMG-boxes linked by a short peptide, which represents a difference with other yeast SOX factors, like Rox1 with its single HMG-box. Yet although Ixr1 binds cisplatin with high affinity, it lacks the phenylalanine that is important for the recognition of cisplatin-DNA adducts by other HMGB proteins.

3.2. Full enhancement of transcription mediated by Ixr1 requires both HMG boxes, which are not functionally interchangeable

ACCEPTED MANUSCRIPT The functionality of the 2 HMG-box domains in transcriptional activation mediated by Ixr1 was first explored, since many HMGB proteins fully recognized as transcriptional regulators contains only one [46-48]. Using qPCR, the effects on

their HMG-box domain region were investigated.

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HEM13 hypoxic activation of a series of Ixr1 mutants with different arrangements of

The Ixr1 mutants studied here included deletions in the HMG-box A

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(Ixr1_HMGAΔ), HMG-box B (Ixr1_HMGBΔ) or both (Ixr1_HMGA/BΔ); as well as chimeras containing 2 copies of HMG-box A (Ixr1_HMGAx2) or 2 copies of HMGbox B (Ixr1_HMGBx2) (Fig. 2A). Yeast cells from an ixr1 null strain were transformed with these constructions. We verified first that mRNA expression from

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these constructions was very similar to those obtained by transformation with a plasmid carrying the wild type IXR1 gene (supplementary material Fig. S3A). Western blot analysis (Fig. S3B) and quantification (Fig. S3A) showed that proteins

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Ixr1_HMGA∆, Ixr1_HMGAx2 or Ixr1_HMGBx2 were expressed at the same levels as the Ixr1 wild type, whereas Ixr1_HMGA/B∆ is very labile, probably due to lower stability of the protein in the absence of the 2 HMG-boxes, which contribute highly to

wild type (Fig. S3B).

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overall folding. Ixr1_HMGB∆ was also expressed, although it only reached 45% of the

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Data on the ability of these Ixr1 variants (Fig.2A) to activate the hypoxic expression of HEM13 show that both HMG-box domains have to be present in the

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Ixr1 protein for full transcriptional activation (Fig. 2B). Moreover, Ixr1_HMGBΔ retains partial activation, attributable to HMG-box A, whereas no activation is observed in the Ixr1_HMGAΔ mutant. Also chimeras, including 2 HMG-box A domains or 2 HMG-

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box B domains, completely lose their capacity to activate hypoxic HEM13 expression. These data show that, in the native protein, each HMG-box has specific and not interchangeable functions that affect recognition of SS targets in vivo, and therefore are required for transcriptional regulation.

3.3. Ixr1 HMG-Box A binds to target DNA sequences with higher affinity than HMG-Box B To elucidate the contribution of the 2 HMG-box domains to DNA binding, equilibrium-binding assays were run with the purified domains. Different Ixr1 protein regions (Supplemental Fig. S1A) containing the HMG-box domains in tandem (tandem-AB, residues 338-510) and both single HMG-box domains A (residues 338439) and B (residues 430-510) were purified to near homogeneity (Supplemental Fig. S1B). Their integrity was also confirmed by Far UV circular dichroism and the secondary structure was calculated (Supplemental Fig. S2). The DNA target

ACCEPTED MANUSCRIPT sequences assayed included B-form DNA duplexes designed from sequences present in the promoter regions of the ROX1 and HEM13 genes, both regulated by Ixr1 (DNAROX1, DNAHEM13_1 and DNAHEM13_2). They are specifically recognized by Ixr1 through these sequences [15, 16], which fit to the general consensus,

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WWCAAW/ WTTGWW, defined for SOX transcriptional factors (Supplemental Fig. S1C); DNAAT duplex used to characterize the structure of the complex of HMG-D D74

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from D. melanogaster; a DNA without specific sequence recognition [25]; and cisplatin- modified DNA as in a previous study with the Ixr1 protein [41]. In a first approach, DNA binding was analyzed by EMSA (Fig. S4). After verifying the interaction, fluorescence anisotropy titrations were carried out to obtain

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more accurate association isotherms of Ixr1 HMG-box domains to different types of DNA duplexes labelled with FAM under different solvent conditions (Table 1; Fig. S5). Affinities calculated by fluorescence anisotropy show that the tandem-AB binds

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to DNAROX1 1.5-2 fold more tightly than single HMG-box A, and up to 7 fold tighter than single HMG-box B. Similar results were obtained with other sequence-specific target sequences such as DNAHEM13_1 or DNAHEM13_2 (Table 1; Fig. S5). Low values

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of association constants for individual HMG-box domains have been found for other HMGB proteins with tandem domains, e.g. yeast Hmo1 [19] or human

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HMGB1 and TFAM proteins [17, 18]. Considering that the isoelectric points calculated for the HMG- box A and HMG-box B are 10.18 and 8.03, respectively, the

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ionic bonds between the positively charged protein domain and the negative charged DNA could to some extent explain the higher affinity measures obtained with HMGbox A than with HMG-box B. Interaction with HMG-box A clearly offers more positive

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areas to interact with ionized phosphates in DNA. Surface charge distribution of both domains for their DNA interaction regions is shown in Fig. 1C. In general, all protein constructs show higher affinities with DNACisplatin than

with B-form DNA duplexes, with some exceptions for HMG-box B (Table 1). For tandem-AB binding to DNACisplatin , the Ka value of 8.33 x 106 M-1 is similar to that obtained by Lippard et al. [41] for the binding of full-length Ixr1 protein to a 92 bp platinated DNA, which indicates that the other regions of the protein do not contribute significantly to binding. It is remarkable that high affinity for DNA-cisplatin adduct binding is reached despite the HMG-box A not containing equivalent to F37 of the human HMGB1, which is essential for this interaction [42]. This evidence opens up new ways of recognising platinated DNA by HMG-box domains.

3.4. ITC assays show that binding of the HMG-box domains to the DNA target in the ROX1 promoter is entropy driven

ACCEPTED MANUSCRIPT Proteins containing HMG-box domains interact usually with the minor groove of DNA, inducing structural effects, such as bending and kinking [3]. In the 2 HMG-box domains of Ixr1, hydrophobic amino acids, flanked by basic residues, are conserved at the strategic positions, producing DNA-contacts (Fig.1), suggesting a similar model

carried out with this protein in support this prediction.

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of interaction. However, structural or thermodynamic approaches have not been

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Characterization of the energetic components that take part in the formation of the protein-nucleic acid complexes shows the nature of the interactions and also aids the differentiation of the binding to the major or the minor groove, since the latter is characterized by positive changes in enthalpy [49], and it is therefore an entropy-

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driven process. To obtain a complete thermodynamic description of the Ixr1-DNA complex formation process from the free components, isothermal calorimetry (ITC)

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was used to measure the binding enthalpies of association, in which the DNAROX1 ligand was titrated into a solution of HMG-box A or HMG-box B. ITC thermographs fitted to a typical calorimetric reaction upon addition of DNA fractions, showing an initial strong heat uptake that decreased when the binding sites on DNA had

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become saturated (Fig. 3). The plot of heat evolved per injection (∆Qi) versus molar ratio is shown in the inset panels of Fig. 3. A non-linear least-squares fit of the

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binding curves to a model with one (type) binding site was successfully applied, confirming the association constant and enthalpy values (Table 2) with an integer

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stoichiometry of ligand-protein close to 1:1 (n ≈ 1) for both HMG-box A and HMGbox B titrations. The difference between the values obtained for KHMGB, the

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association constant of HMG-box B calculated by ITC (Fig. 3B) or by FA, is attributable to the unfeasibility of reaching saturation in the ITC experiments due to the low affinity of the ligand for this site in absence of the other. For both ITC titrations, enthalpy values determined for HMG-box A and HMG-box B are positive (30.8 and 32.2 kJ/mol, respectively, Table 2), supporting Ixr1 binding to the minor DNA groove [49]. Titration of DNAROX1 with the tandem-AB construct (residues 338-510) gave the calculation of the macroscopic binding constants, defined as free-model parameters [26]; the results obtained show a number of interesting features that suggest a binding model, shown along with all the calculated binding constants in supplemental material (Fig. S6). Binding isotherms for the interaction of tandem-AB with DNAROX1 had a ‘biphasic’ pattern, with additional heat effects at high saturation, corresponding with the sequential binding of the 2 HMG-box domains within this construct (Fig. 3C). Since no previous information has helped describe a model of interaction of the 2 HMG-box domains of Ixr1 with the target sequences that are implicated in transcriptional

ACCEPTED MANUSCRIPT activation mediated by this protein, we analysed the data in depth. As in other proteins with 2 or more HMG-box domains, DNA binding to the first box might affect the binding to the other/s [17, 19, 28]. In our experimental approach, fitting to a model with 2 identical binding sites was discarded because clearly the binding of one of the HMG-

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box domains excluded the binding of the other one to the same ligand region. Besides, the values of heat released due to DNA binding to individual HMG-box

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domains alone are between those of ∆H1 and ∆H2 in the tandem (Table 2), suggesting that the model regarding independent binding is unlikely. Therefore, analysing the tandem-AB binding to linear DNAROX1 using a model with 2 sequential binding sites (see Materials and Methods) yielded the stepwise (Kj) and associated

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enthalpy changes of 2.05 x 107 M-1 (K1) and 7.60 kJ/mol (∆H1), and 2.91 x 105 M-1 (K2) and 89. 5 kJ/mol (∆H2) for each of the 2 HMG-box domains. The calculated

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overall association of β2 was 5.97 x 1012 M-1 and associated enthalpy change of 97.1 kJ/mol. The model also helped the calculation of microscopic constants KHMGA, HMGB

and KHMGB, HMGA. These values, obtained by ITC, as well as previous information

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obtained by fluorescence anisotropy titrations (see above section), suggest that binding to the HMG-box A corresponds with the first binding event, and binding to HMG-box B with the second event. The value (470) obtained for the microscopic

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cooperative constant C12 deduced from the ITC study as well as those of Hill slope (n cooperativity.

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= 2.03±0.16) obtained in FA experiments support the existence of positive

In summary, the analysis of our results is compatible with Ixr1 binding to the

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minor grove of the B-form of DNA, and with a sequential model of interaction, in which HMG-box A is the first event and favours HMG-box B binding by a positive cooperative effect.

3.5. The HMG-Box A has a higher contribution to DNA bending than the HMG-Box B

DNA bending by HMGB proteins provides a mechanism for transcriptional regulation [4]. We measured the effect of the interactions with HMG-box A and HMG-box B domains upon DNAROX1 bending by FRET. Since spectral overlap exists between FAM emission and TAMRA excitation, a decrease in the fluorescence emission of FAM at 520 nm, and an increase in the fluorescence emission of TAMRA at 580 nm, occur when the end-to-end distance of the DNAROX1, and concomitantly the distance between the fluorophores, decreases. DNAROX1 bending by binding to the HMG-box domains decreases the distance between FAM and TAMRA, and thus

ACCEPTED MANUSCRIPT increases the FRET effect. We calculated the bending angles corresponding to the binding of Ixr1 HMG-box A and HMG-box B to DNA DNAROX1 by the method of Malarkey et al. [28]. Fig. 4 shows FRET effect plots, and processed data indicate that HMG-box A binding produces a bending of 62±3 in linear DNA, and HMG-box B

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binding bends DNA in only 40±8. Therefore, cooperative sequential binding 0 expected for tandem-AB will produce a total bend angle of 99 ± 2 , which is in the

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range previously reported for other HMGB proteins [5, 6].

3.6. Thermodynamic footprint of HMG-box A, but not that of HMG-box B, changes in the interaction with platinated DNA in comparison with the B-form of DNA.

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Binding of tandem-AB to DNA Cisplatin produces a ‘biphasic’ pattern, as with other non-platinated DNA targets (Fig. 5C), but in this case it was not possible to fit

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the data to a sequential model with positive cooperativity (data not shown). A striking difference between both HMG-box domains of Ixr1 has been the enthalpy sign of the binding to DNACisplatin: ΔH = -39.9 kJ/mol for HMG-box A and ΔH = 38.4 kJ/mol

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with the HMG-box B (Fig.5A and B; Table 3). These differences might indicate different entropy/enthalpy compensation mechanisms in binding reactions between both individual HMG-box domains to platinated DNA, and/or binding to different

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regions of the DNACisplatin molecule. Entropy and enthalpy are highly influenced by the numerous water molecules released during binding reactions [56], and these effects

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must also be considered in any explanation. Use of free or platinated DNA in the binding reactions also affects the number

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of ionic contacts formed between the target DNA and the protein. According to the counter-ion condensation concept [25, 50-52] fluorescence anisotropy titrations at increasing salt concentrations helps to calculate the number of ionic contacts (Z) formed between DNA and protein. Using this procedure, binding reactions between the tandem-AB, HMG-box A or HMG-box B and different DNA targets were carried at different KCl concentrations of from 100 to 400 mM. This (Fig. S7) gave the calculated number of ionic contacts (Z) formed between the HMG domains and the phosphate groups of DNA (Fig. S8; Table S2). Z values obtained for binding of HMG-box A to the assayed B-forms of DNA are in good agreement with previous data obtained for individual HMG- box domains without flanking tails, as seen with human LEF1 or HMG-D from D. melanogaster [50], and ranging from 5.20 (HMGbox A+DNAHEM13_1 complex) to 6.34 (HMG-box A+DNATA complex). The fact that the Z numbers were closer to net charge of the domain rather than to total positive charges might be interpreted, as in previous studies carried with other HMGB proteins [25, 50, 52-55] as a consequence of the presence of a substantial number of

ACCEPTED MANUSCRIPT internal salt links on the surface of the HMG-box domains, especially attributed to helix 3 in the HMG fold, i.e. away from DNA interaction. It is noteworthy that the binding of the tandem-AB and HMG-box A to DNACispatin releases ≈9 and ≈8 counter-ions, respectively (Table S2), a value higher than that calculated for binding

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to the B form of DNA. The extra salt links could explain the higher affinity of the

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platinated DNA in comparison to B form DNA.

3.7. Calculation of the non-electrostatic component of ∆Go in the interaction of HMG-box A with different DNA sequences shows that interaction is not specific of

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sequence.

For a better understanding of the interactions between the DNA binding

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domains present in Ixr1 protein and their DNA targets, separation of the overall binding energy into its electrostatic and non-electrostatic components is required. In general, the electrostatic component (el) of the binding energy, which results from

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ionic and polar interactions with the DNA phosphate groups, predominates over the non-electrostatic (nel) for both SS and NSS types of HMG-boxes [25, 50, 51]. Furthermore, the electrostatic component is in any case independent on the

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sequence of target DNA. Specificity depends uniquely on the non-electrostatic component, and therefore this component varies with the DNA sequence, being

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higher for the more perfect target [52]. The method used to split the measured Gibbs’ energies of binding in the electrostatic and non-electrostatic components (ΔGa= ΔGanel + ΔGael) is based on

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the fact that at higher salt concentrations (≈1 M KCl), the electrostatic part vanishes (log[KCl] = 0), and ΔGa = -RTLn(Ka) corresponds to the non-electrostatic part of the Gibbs’ energy of complex formation. ΔG ael component is entirely entropic [50]; thus the entropy factor of the non-electrostatic component (TΔS anel) can be derived by subtracting the non-electrostatic Gibbs’ energy (ΔGanel) from the total enthalpy of association (ΔH a) (TΔSanel= ΔHa - ΔGanel) [52]. Analysing the interactions of HMG-Box A with different DNA ligands, the split in the measured Gibbs’ energies of binding in the electrostatic and nonelectrostatic components shows that the non-electrostatic component has minor changes when compared with sequence-specific targets (DNAROX1, DNAHEM13_1, DNAHEM13_2)and a non-specific DNA sequence (DNATA) (Fig. 6). Therefore the interaction of the HMG-Box A from Ixr1 with DNA is classified as NSS by this experimental approach [50], which is in agreement with data indicating that HMG-box A from Ixr1 aligns more closely with a HMG-Box domain classified as NSS [16]. In the

ACCEPTED MANUSCRIPT case of titrations with HMG-Box B, saturation was not reached due to low affinity (Fig. S7), and therefore no conclusion could be drawn about the HMG-Box B.

3. 8. Configuration of the HMG tandem A/B domain in Ixr1 is required to stabilize

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HMG-box B folding

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A striking difference in the organization of HMG-box A and HMG-box B domains in Ixr1 compared to other HMGB proteins with 2 HMG-box domains is the short linker region between them. To determine whether the correct folding of the 2 in tandem HMG-box domains of Ixr1 is achieved independently in solution, we

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compared 2D NMR1 H - 1 5 N HSQC spectra of single HMG-box A and HMG-box B with those of tandem-AB. The cross- peaks in the 1H-15N HSQC spectrum of tandem-AB

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were broader than cross-peaks in the 2D spectrum of single HMG-box domains, indicating that the tumbling of the tandem-AB is slower than individual ones (Fig. 7A and Fig. S9). The addition of linear DNA to form tandem-AB+DNAROX1 complex did not improve the signal spectra (data not shown). Although tandem-AB peaks (black

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in Fig. 7A) can generally be associated with the corresponding HMG-box A (blue in Fig. 7A) and HMG-box B (orange in Fig. 7A) peaks, there are some differences

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in chemical shift to indicate that the environment is different in the tandem-AB and individual domains. In the case of HMG-box A, 2D spectrum peaks fit well with the 2D

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spectrum of tandem-AB counterpart, indicating similar folding of the HMG-box A domain in the 2 peptides. Nevertheless, the 1H-15N HSQC spectrum with HMG-box B domain shows extra peaks in the 8.0-8.5 ppm region that have no counterparts in

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the tandem-AB domain spectrum, implying conformer variations or uncompleted folding of HMG-box B (Fig. 7B). Counting the non-amine peaks in 1H-15N HSQC spectrum of HMG-box B domain, the sum reaches >100 peaks, which far exceeds the number of expected peaks (71). This supports the assertion that folding of the free HMG-box B domain in solution is more than one conformation, or that the HMG-box B is not completely folded and its stabilization in the most stable conformer, or its complete folding requires the proximity of the HMG-box A domain.

3.9. HMG-box B binding contributes to the stabilization of the protein-DNA complex

Far UV-CD was used to determine changes in the secondary structure profiles after DNA binding (Fig. S2). After adding SS DNAROX1, only the tandem-AB protein showed a 7% increase in α-helical content (Table 4); however, individual HMG-box domains were unchanged in their circular dichroism profiles (Fig. S2). This

ACCEPTED MANUSCRIPT indicates that both HMG boxes are simultaneously needed for the dynamic transition from the unbound to the bound state with this DNA, a transition that stabilizes tandem folding. We have characterized the proteins and protein-DNA complexes in terms of

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thermal stability, analysing the temperature dependence of the molar ellipticity at 222 nm. There was a decrease in the negative values of molar ellipticity at 222 nm over

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the range from 20 to 80oC towards zero, corresponding to a completely unfolded state. Besides this major transition due to the anticipated unfolding by denaturation at high temperatures, the ellipticity also changes even at lower temperatures (5 to 200C). In o this sense, Fig. 7C (continuous lines) shows that the ellipticity change below 20 C is

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especially pronounced in the case of HMG-box B, indicating that B is more labile in solution. The first derivative of the circular dichroism melting curves (Fig. 7C inset)

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shows the melting temperatures of HMG-box A and B of Ixr1 are very similar (Table 3). A single transition was seen in the case of the tandem-AB, suggesting that both HMGbox A and HMG-box B unfold at similar temperatures, or unfold in a cooperative way (Fig. 7C). The melting temperatures calculated by Differential Scanning Fluorimetry

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confirm these results (Fig. S10). When duplex DNAROX1 was added (Fig. 7B, dashed lines), thermal stability improved (results summarized in Table 4). In the case of the

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HMG-box B, Tm increased 4oC, whereas the HMG-box A increased by only 0.5ºC . When both HMG-box domains were in tandem, the increment was 3ºC in comparison

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with the individual domains, even without a DNA ligand, which supports the dependence of HMG-box B on HMG-box A’s proximity in order to reach a more stable

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folding state (Table 4).

The presence of 2 tryptophan amino acids situated in similar positions of both HMG-box A (W399 and W418) and HMG-box B (W472 and W494), as well as similar wedge distances between aromatic amino acids based on structural homologue models, helps to compare their intrinsic fluorescence spectra after excitation at 280 nm so as to see any differences in the exposure of these amino acids to the solvent. The HMG- box B has a higher fluorescence (i.e. exposed tryptophan) and the binding of the DNA produces broader changes (21% in HMG-box A and 27% in HMG-box B), which means broader rearrangements with HMG-box B (Fig. 7D; Table 4). These data are in accord with measurements on thermal stability, and also argue in favour of an important contribution of the HMG-box B in stabilizing the protein-DNA complex.

4. Discussion In S. cerevisiae, ROX1 and IXR1 are considered SOX genes encoding

ACCEPTED MANUSCRIPT transcription factors involved in adaptation to aerobic-hypoxic conditions [7]. HMGB proteins, Rox1 and Ixr1, are only expressed at high levels under specific conditions Rox1 during normoxia and Ixr1 during hypoxia - and they control the expression of genes needed to adapt to these particular environments [9, 15, 16]. Although

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mechanisms that control transcriptional regulation mediated by Rox1 has been extensively studied [57-60], those sustaining Ixr1 functions in transcription or DNA

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repair are less known. We therefore analysed the significance of the 2 HMG-boxes present in Ixr1 in relation to DNA binding properties related to transcriptional regulation or recognition of cisplatin-DNA adducts.

HMGB proteins with transcriptional regulatory functions usually have only one

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SS HMG-box domain [46-48, 58], with rare exceptions like TFAM [34, 35, 61]. Alignment of the 2 HMG-box domains of Ixr1 with other proteins with 2 in tandem

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HMG-box domains (Fig. 1A) shows typical characteristics of NSS for HMG-boxes A and B, i.e. the presence of 2 hydrophobic amino acids, putative DNA-intercalating residues, one at the beginning of the first alpha-helix and a second one at the amino-terminal side of the second alpha-helix in the HMG domains. This is in

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contrast with previous observations regarding SS HMG-box domains, in which this second position is occupied by a polar residue making specific contact with nitrogen

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bases through hydrogen bonds, and contributing to sequence-specific recognition [52]. Indeed, the results in Table 2 indicate that, when assayed separately, both

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HMG-box domains have a thermodynamic signature corresponding to that previously defined for the NSS family [50], with ΔH values of 30.8 and 32.7 kJ/mol, for HMG-box A and HMG-box B, respectively. Calculation of the non-electrostatic

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component of the binding energy is also in accordance with the consideration that HMG-box A is NSS (Fig. 6), and no conclusion could be drawn about the HMG-box B. This was intriguing, since previous evidence using in vitro competition assays confirmed that purified Ixr1 binds specifically to SS DNA targets [15, 16]. We have proved that mutant Ixr1 derivatives with only one HMG-box (A or B) or carrying 2 identical A or B boxes are inefficient in transcriptional activation of the HEM13 target (Fig. 2B), which suggests that the co-existence of two different HMG-boxes A and B in Ixr1 in vivo is a requirement for the recognition of specific regulatory sequences in the promoters. Therefore, a simple model assuming that one HMG-box allows NSS and the other allows SS recognition can be discarded. The question then arises as to why the conformation of the tandem-AB in the Ixr1 protein is important for SS recognition. Our data (microscopic cooperative constants deduced from the model presented in Figure S6 and Hill coefficient calculated from FA experiments) indicate the existence of positive cooperativity, in

ACCEPTED MANUSCRIPT which the binding and bending processes of the first HMG-box domain assist in the binding and bending of the second one, as reported for other HMGB proteins such as TFAM [18], HMGB1 [62] and Hmo1 [63]. This sequential model of interaction is functionally important to reach finally an adequate bend angle, since only the Ixr1

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with the native tandem-AB configuration produces full transcriptional activation, as previously reported in this work. Otherwise, proteins with tandem repetitions of HMG-

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box A or HMG-box B domains behave as the ixr1∆ null strain (Fig. 2). Total deviation from the axis of the double helix of DNA in the DNA-protein complex may be important in ensuring interaction of the transcriptional factor Ixr1 with the general transcriptional machinery. Alternatively, due to the short and rigid connection

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between HMG-box A and B (limited to only 5 amino acids), this bending could be a consequence of necessary rearrangements to fit more precisely the intercalating

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residues of the DNA binding domains inside the double helix of the target sequence. NMR data (Fig. 7A, 7B and S9), suggesting that more stable folding of the HMGbox B can be obtained when the HMG-box A is present in the construct. One can speculate that differences in sequence specificity recognition, when the HMG-box B

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is or is not in tandem with HMG-box A in the experimental constructs, are due to incomplete folding when the HMG-box B is alone. Remarkably, although the HMG-

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box B has a lower affinity for DNA than the HMG-box A, its role in complex formation is crucial in increasing stability, as deduced from CD-melting curves (Fig. 7C), or by

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measuring intrinsic fluorescence changes due to specific Trp residues masking after forming complexes with DNA (Fig. 7D). Regarding the role of Ixr1 in DNA repair, it is important to know how

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transcription targets and cisplatin adducts are differentially recognized by the same protein, leading first to interaction with the basal transcriptional machinery [7-8], and secondly with the proteins involved in avoiding DNA repair [13]. Our data show that the affinity of the constructions (single or in tandem HMG-box domains) for platinated DNA is 2-3 fold higher than the affinity for target sequences in transcriptionally regulated promoters (Table 1). Clear differences exist in the thermodynamic signature of the 2 HMG boxes of Ixr1 whether binding to DNAROX1 or DNACisplatin (Fig. 8). The binding of Ixr1 HMG-box A to platinated DNA is enthalpy-driven (Fig. 8A); this thermodynamic characteristic of the HMG-box binding to platinated DNA was predicted from a theoretical approximation [64], but to our knowledge this is the first experimental data to support this prediction. On the other hand, binding of HMG-box B is entropy-driven (Fig. 8B), whereas the union in the tandem-AB to the first site (assuming that it is HMG-box A) is enthalpy driven, and to the second is entropy driven. The lower values of the entropy energetic component when HMG-box A binds

ACCEPTED MANUSCRIPT to platinated DNA could be attributed to the pre-bend state of the DNA, due to the intra-strand crosslink with cisplatin, or to different architecture of the water molecules displaced from the minor grove by the binding [38, 51, 64]. In the tandem-AB, the high and positive values obtained for the enthalpy component of binding to site 2, and

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consequently from the calculated entropy-component, might reflect the fact that these values are highly affected by protein refolding or binding to adjacent DNA regions

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close to the cisplatin adduct.

In summary, we propose a model for Ixr1 interaction with SS or NSS DNA targets in transcriptional activation or to cisplatin-DNA adducts (Fig. 9). This model also explains the results obtained regarding the functional capacity of different Ixr1

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deletions and chimeras to activate the HEM13 gene reporter during hypoxia (Fig. 2). Our data on complexes formed between tandem-AB and DNAROX1, indicate that

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the DNA first binds to the HMG-box A with higher DNA binding affinity, producing a bending angle of 62o in linear DNA. This event might produce a conformational change, propagated to the HMG-box B, along with a higher probability of

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contacting with specific DNA, that enhances its DNA binding affinity. The second event of binding through HMG-box B would result in an additional bend angle up to 99o. In this model, the interaction through the HMG-box A allows binding to a NSS

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DNA sequence without the participation of HMG-box B; but the resulting pre-complex would not be fully stable, would not reach the total DNA bending, and would

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dissociate in a high percent of tentative trials without producing functional interaction with the general transcriptional machinery; therefore it would not cause transcriptional

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activation (Fig. 9A). This is in accord with the previous finding that HMG proteins are very dynamic in their interactions with DNA and move quickly along it [4, 65]. This model explains fully our results showing that deletion of HMG-box B significantly diminishes the hypoxic activation of the HEM13 reporter. However, when interaction is produced with one specific DNA target (Fig. 9B), both HMG-box A and HMG-box B would participate in the formation of the complex with DNA, the latter definitively contributing to total DNA bending and complex stabilization. Deletion of HMG-box A, or the organization of domains in the chimera carrying 2 repetitive HMG-box B domains, probably produces no fruitful interaction due to the lower affinity of HMG-box B for DNA. The chimera carrying 2 repetitive HMG-box A domains is also unproductive in HEM13 hypoxic activation, which could be attributed to over DNA-bending and/or insufficient complex stabilization in the absence of HMG-box B. Finally, irrespective of the molecular causes that might generate the differences in thermodynamic pattern reported for Ixr1 binding to

ACCEPTED MANUSCRIPT DNACisplatin (Fig. 8), binding of Ixr1 HMG-box domains to cisplatin adducts presumably generates a final structure unrecognized by the machinery of transcription (Fig. 9C), which would explain the dual role of Ixr1 in these 2 processes. Transcription, DNA damage, and repair might be physically and functionally

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intertwined [1]. Transcription is regarded as a risky task that might cause DNA damage; conversely DNA damage can activate several transcription events [1].

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Hypoxia is a signal of stress for yeast that also causes DNA damage [66, 67], the cell adapting to this stressor with a wide reorganization of its transcription pattern [60]. Therefore, the co-existence of the 2 HMG domains in Ixr1, and the consequent possibility of differentially binding to transcription or repair-prone targets could be a

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beneficial trait for the evolution of this protein in parallel with the evolution of species of yeasts. This is particularly interesting considering any changes needed in adapting

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yeast cells from a strictly anaerobic metabolism to being facultative anaerobes that can survive in niches with different oxygen levels [68, 69].

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6. Conclusion

In tandem arrangement of the 2 HMG-boxes in Ixr1 is clearly necessary to

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form a stable complex between specific regulatory sequences and the protein that allows transcriptional activation in vivo. The formation of this complex adjusts to a

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sequential positive cooperative model, with binding to both HMG-box A and B being entropy driven. HMG-box A has a higher affinity than HMG-box B for DNA targets,

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but does not determine sequence specificity. HMG-box B binding, although of lower affinity, mostly contributes to complex stabilization. Binding to platinated DNA does not fit with a cooperative model, is enthalpy

driven for the HMG-box A, and entropy driven for the HMG-box B. We show experimentally for the first time that HMG-box binding to different DNA structures is associated with predictable thermodynamic differences. Although the HMG-box A does not recognize specific sequences, our data suggest that the nature of its interaction with different DNA structures might regulate interaction with the HMG-box B in the final organization of the complex. Thus, functional specialization of the 2 HMG-box domains allows differential recognition of SS regulatory sequences or cisplatin-DNA adducts, and contributes to the dual function of Ixr1 regulating transcription and DNA repair.

7. Acknowledgements This research was supported by grant BFU2009-08854 from MICINN (Spain),

ACCEPTED MANUSCRIPT co-financed by FEDER (CEE). General support to the laboratory during 2012-16 was funded by Xunta de Galicia (Consolidación D.O.G. 10-10-2012. Contract Number: 2012/118) and co-financed by FEDER. A V’s salary was funded by a Predoctoral fellowship (I2C program of 2011) from Xunta de Galicia. A.V’s short stay in the

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Department of Biochemistry of the University of Cambridge was funded by UDCInditex Fellowship (2014). We thank K Stott and J O Thomas for their contribution

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and advice during this stay. ITC experiments were carried out at the Elemental Analysis Unit of the Santiago de Compostela University. CDs were assayed at the Molecular Spectroscopy Unit of the A Coruña University. pKLSL150 plasmid was kindly donated by J M Mancheño (Institute of Physical Chemistry Rocasolano-CSIC,

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Madrid, Spain). The English text was edited by Biomedes

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(http://www.biomedes.co.uk/).

8. Authors’ contributions

MEC and AVV conceived and coordinated the study. AVV designed and

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performed the experiments in collaboration with MLM (EMSA experiments) RFL, ARD and MB (CD and ITC experiments). NMR experiments were carried out during the

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stay of AVV at the Department of Biochemistry of the University of Cambridge. AVV worked with MEC to write the draft of the manuscript. All authors read the draft,

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contributed with useful discussions and approved its final content.

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9. References

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ACCEPTED MANUSCRIPT domain protein, to cisplatin-DNA adducts in vitro and in vivo, Biochemistry, 35 (1996) 6089-6099. [42] U.M. Ohndorf, M.A. Rould, Q. He, C.O. Pabo, S.J. Lippard, Basis for

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Analyses of Anaerobically Induced Genes in Saccharomyces cerevisiae: Functional Roles of Rox1 and Other Factors in Mediating the Anoxic Response,

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solution structure of the human SRY-DNA complex, Cell, 81 (1995) 705-714. [48] L.S. Whitfield, R. Lovell-Badge, P.N. Goodfellow, Rapid sequence evolution of the mammalian sex-determining gene SRY, Nature, 364 (1993) 713-715. [49] P.L. Privalov, A.I. Dragan, C. Crane-Robinson, The cost of DNA bending, Trends Biochem. Sci., 34 (2009) 464-470. [50] A.I. Dragan, C.M. Read, E.N. Makeyeva, E.I. Milgotina, M.E. Churchill, C. Crane-Robinson, P.L. Privalov, DNA binding and bending by HMG boxes: energetic determinants of specificity, J. Mol. Biol., 343 (2004) 371-393. [51] P.L. Privalov, A.I. Dragan, C. Crane-Robinson, K.J. Breslauer, D.P. Remeta, C.A. Minetti, What drives proteins into the major or minor grooves of DNA?, J. Mol. Biol., 365 (2007) 1-9. [52] P.L. Privalov, A.I. Dragan, C. Crane-Robinson, Interpreting protein/DNA interactions: distinguishing specific from non-specific and electrostatic from non-

ACCEPTED MANUSCRIPT electrostatic components, Nucleic Acids Res., 39 (2011) 2483-2491. [53] J.J. Love, X. Li, D.A. Case, K. Giese, R. Crosschedl, P.E. Wright, Structural basis for DNA bending by the architectural transcription factor LEF-1, Nature,

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376 (1995) 791-795. [54] F.V. Murphy, R.M. Sweet, M.E. Churchill, The structure of a chromosomal

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high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition, EMBO J., 18 (1999) 6610-6618.

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Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation, J. Mol. Biol., 312 (2001) 481-

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ACCEPTED MANUSCRIPT looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin, Nucleic Acids Res., 42 (2014) 8996-9004. [64] T.H. Nguyen, G. Rossetti, F. Arnesano, E. Ippoliti, G. Natile, P. Carloni,

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Molecular Recognition of Platinated DNA from Chromosomal HMGB1, J. Chem. Theory Comput., 10 (2014) 3578-3584.

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Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes, J. Biol. Chem., 277 (2002) 34773-34784.

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LEGEND TO THE FIGURES

Figure 1. Characteristics of the HMG-box domains of Saccharomyces cerevisiae

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Ixr1 (A).

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In tandem HMG-box domains of Ixr1 from S. cerevisiae (Uniprot code P33417) are aligned with other proteins with in tandem HMG-boxes: TFAM from Homo sapiens (Uniprot code Q00059), Abf2 from S. cerevisiae (Uniprot code Q02486), DSP1 from D. melanogaster (Uniprot code Q24537), HMGB1 (Uniprot code P09429), HMGB2

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(Uniprot code P26583), HMGB3 (Uniprot code O15347) and HMGB4 (Uniprot code Q8WW32) from H. sapiens. Intercalating residues are highlighted in purple and green (from the primary site and the secondary site, respectively). Phenylalanine intercalating

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residues are highlighted in yellow, and conserved residues are highlighted in black. The first HMG-box domain (HMG-box A) is framed inside the blue box and second (HMG-box B) inside the orange box. Linker regions are framed in grey. Residue

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numbering follows the original positions in the full-length Ixr1 protein (upper-side numbers) or in the other proteins used in the alignment (right-side numbers). (B)

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Schematic representation of the L-shaped domain structure of HMG-box A (blue) and HMG-box B (orange), showing the amino acids that are candidates to be DNA intercalators, F369 and I442 in purple sticks, and V388 and L461 in green sticks. HMG-

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box A and HMG-box B were modeled by Phyre2 server using Tox2 protein from Mus musculus (PDB 2CO9) and HMGB1 from human (PDB 2E6O) as templates. (C)

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Surface charge distribution in HMG-box A and HMG-box B domains. Positive charges are blue and negative red. Pink arrows indicate the concave surface of binding with DNA.

Figure 2. Expression analysis of the HEM13 gene in different Ixr1 mutant strains of S. cerevisiae during hypoxic conditions. (A) HMG-box domain configuration of the different Ixr1 mutants tested for HEM13 transcriptional activation (B) HEM13 expression is represented relative to the expression of the reference gene TAF10, and normalized to the levels in normoxic cultures. Two different biological replica and 2 technical replicates of each were done and their values averaged. The nomenclature of the different Ixr1 mutants is described in the text. Significance thresholds (*p<0.05; **p<0.01) are indicated in red, whether in reference to the ixr1∆ strain (represented in the second bar) or in black in reference to the ixr1∆ strain transformed with Ycplac33IXR1 construction (represented in the first bar).

ACCEPTED MANUSCRIPT Figure 3. Isothermal titration assays with DNAROX1. Thermodynamic analyses of (A) HMG-box A (blue), (B) HMG-box B (orange) and (C) tandem-AB (black) in the interaction with to DNAROX1. ITC thermograms show the raw heats absorbed after injecting target DNA (syringe) into protein solution (cell). Inset plots show the integrated

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absorbed heats with respect to time with the heat of mixing subtracted and against

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protein:DNA molar ratio.

Figure 4. Förster resonance energy transfer assays. Plots of the FRET effect on DNA bending with the doubled-labeled 20 bp DNAROX1 after titration with (A) HMG-box A (blue), (B) HMG-box B (orange) and (C) tandem-AB (black). Data points are the

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average of 3 independent experiments, with error bars representing standard deviations. Inset plots show changes in fluorescence spectra from high (dark color) to low concentrations (light color) of binding protein. The peak at 520 nm is the

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fluorescence of FAM (the donor) and the peak at 580 nm the fluorescence of TAMRA (the acceptor).

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Figure 5. Isothermal titration assays with platinated DNA. Thermodynamic analyses of (A) HMG-box A (blue), (B) HMG-box B (orange) and (C) tandem-AB (black) to DNACisplatin. ITC thermograms show the raw heats absorbed from injecting

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target DNA (syringe) into protein solution (cell). Inset plots show the integrated absorbed heats with respect to time with the heat of mixing subtracted and against

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protein:DNA molar ratio.

Figure 6. Electrostatic and non-electrostatic components in the interactions of

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Ixr1 HMG-boxes with SS (ROX1, HEM13_1, HEM13_2) and NSS (TA) DNAs. The electrostatic (in blue) and non-electrostatic (in lime) components of the total Gibbs’ free energy of binding of HMG-box A with different linear DNA duplexes, according to the calculations described in the text. Figure 7. Stability and folding state of the in tandem and individual HMG-box domains of Ixr1 in solution. (A) 2D 1H-15N HSQC spectra of free proteins used in this study. The plot includes the superposed data of Tandem-AB (black), HMG-box A (blue) and HMG-box B (orange) domains of Ixr1 protein. (B) Close-up view of in the 8-7.75 ppm (1H) emphasising the lack of overlap between tandem-AB and HMG-box B spectra. (C) CD melting curve of tandem-AB (black), HMG-box A (blue) or HMG-box B (orange) in the absence (continuous lines) or the presence of DNAROX1 (dashed lines), and starting from 10oC and finishing at 80oC. Inset shows first derivative of CD melting curves as a function of time for Tm calculation. (D) Intrinsic fluorescence spectra of

ACCEPTED MANUSCRIPT HMG-box A (blue) and HMG-box B (orange) in the absence (continuous lines) or the presence of DNAROX1 (dashed lines) after excitation at 280 nm. Figure 8. Differences in the enthalpic and entropic contributions to the non-

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electrostatic Gibbs’ energy of (A) HMG-box A and (B) HMG-box B binding to DNAROX1 or DNACisplatin. The non-electrostatic Gibbs’ energies of binding are shown by

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the blue bars, the enthalpies by red bars and the entropic factors by yellow bars. Figure 9. Hypothetical model of Ixr1 binding to different targets. This model helps to explain the differential role of the HMG-box A and the HMG-box B in the recognition of (A) NSS targets, (B) SS targets, (C) Cisplatin-DNA adducts, and their implications in

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the cellular function of Ixr1 (see text for a detailed description).

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Table 1

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Affinity constants calculated by Fluorescence Anisotropy in the interactions of HMGboxes and DNA targets. Ka (M-1) tandem-AB

Ka (M-1) HMG-box A

Ka (M-1) HMG-box B

DNAROX1

1.69 x 106 ± 0.03

1.30 x 106 ± 0.04

2.30 x 105 ± 0.46

DNAHEM13_1

1.96 x 106 ± 0.04

1.19 x 106 ± 0.02

1.19 x 105 ± 1.86

DNAHEM13_2

3.23 x 106 ± 0.02

1.59 x 106 ± 0.05

1.18 x 105 ± 1.50

DNATA

3.57 x 106 ± 0.02

1.67 x 106 ± 0.06

1.68 x 105 ± 1.58

DNACisplatin

8.33 x 106 ± 0.01

4.17 x 106 ± 0.03

1.37 x 105 ± 1.52

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DNA

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Table 2

Thermodynamic characteristics of HMG-box domains binding to DNAROX1.

(Chi2/DoF = 8.42 x 105)

-41.74 ± 0.89 (K1) -31.17 ± 0.12 (K2)

7.60 ± 3.13 (from K1)

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Tandem 2.05 AB x 107 ± 0.92 (K1)

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Stoichiometry ΔG ΔH TΔS ΔG TΔS (Chi2/DoF) (kJ/mol)a (kJ/mol)a (kJ/mol)a (kJ/mol)b (kJ/mol)b,c

Ka (M1 a )

89.45 ± 5.36 (from K2)

49.62 (from K1)

-35.53 ± 0.04

123.68 (from K2)

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Protein

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2.91 x 105 ± 0.14 (K2) 5.29 x 105 ± 0.27

1.09 ± 0.04 (Chi2/DoF = 1.18 x 105)

-32.65 ± 0.13

30.79 ± 1.44

64.46

-34.88 ± 0.08

65.67

HMGbox B

2.40 x 104 ± 3.78

0.63 ± 0.42 (Chi2/DoF = 2.25 x 105)

-24.99 ± 2.34

32.22 ± 14.37

58.22

-30.59 ± 0.45

62.81

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HMGbox A

a

Obtained by ITC (Isothermal Titration Calorimetry) Obtained by FA (Fluorescence Anisotropy)

b

Ka: Affinity constant Gibbs energy (ΔG) were obtained with the equation ΔG = -RTLn(Ka), where R= 8.314472 J L-1 mol-1; and T= 298 K c Enthropy (TΔS) were obtained from the equation: TΔS= ΔH - ΔG

Table 3

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Thermodynamic characteristics of HMG-box domains binding to DNACisplatin.

3

3.57 x 10 ± 1.34 (K2)

3.40 x 106 ± 0.60

HMGbox B

7.25 x 104 ± 0.46

-

-37.63 ± 1.05 (K1)

(Chi2/Do F = 4.26 x 106)

-20.27 ± 0.79 (K2)

-60.69 ± 6.86 (from K1) 772.78 ± 95.36 (from K2)

TΔS (kJ/m ol)a

ΔG (kJ/mol)

30.79 (from K1)

-39.48 ± 0.01

-

b

794.1 9 (from K2)

-39.93 ± 0.79

-4.01

-37.77 ± 0.02

-2.16

0.80 ± 0.34 (Chi2/Do F = 1.86 x 105)

-27.73 ± 0.15

38.35 ± 4.78

67.32

-29.31 ± 1.85

65.19

a

Obtained by ITC (Isothermal Titration Calorimetry) Obtained by FA (Fluorescence Anisotropy) Ka: Affinity constant b

Gibbs energy (ΔG) were obtained with the equation ΔG = -RTLn(Ka), where R= 8.314472 J L-1 mol-1; and T= 298 K c

TΔS (kJ/mo l)b,c

-37.26 ± 0.41

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ΔH (kJ/mol)a

0.99 ± 0.01 (Chi2/Do F = 1.06 x 105)

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HMGbox A

ΔG (kJ/mol)a

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3.95 x 106 ± 2.07 (K1)

Stoichio metry (Chi2/Do F)

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Tande m-AB

Ka (M-1)a

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Enthropy (TΔS) were obtained from the equation: TΔS= ΔH - ΔG

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Stability analyses.

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Table 4 α-helix content (%)

Tm (oC)

Intrinsic fluorescence (% reduction)a

Tandem-AB

69

46.5

-

Tandem-AB + DNAROX1 complex

76

47.5

-

48

43

-

48

43.5

21.34

69

43.5

-

69

47.5

26.96

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Protein

HMG-box A HMG-box A + DNA

complex

HMG-box B ROX1

HMG-box B + DNA

complex

a

MA

ROX1

AC

CE

PT

ED

Fluorescence counts of 1µM protein:DNA pulse at 280 nm.

ROX1

complex at 335 nm after an excitation

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Highlights

PT

HMG-box A and HMG-box B of Ixr1 have specialized functions in the recognition of DNA at regulated promoters

SC RI

HMG-box A binds to regulatory sequences with higher affinity and produces higher DNA bending than HMG-box B

NU

In tandem configuration of HMG-box A and HMG-Box B influences folding, favors DNA binding and is necessary for specific DNA recognition

AC

CE

PT

ED

MA

Binding of HMG-box A to specific regulatory sequences of DNA or cisplatin adducts is thermodynamically distinguishable