TONSL/TONSUKU binding to histone H3.1 is required for DNA repair

Data files

Sep 17, 2026 version files 3.22 GB

README.md

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TONSL_HDXMS_Dataset.zip

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Abstract

In plants, TONSUKU (TSK) regulates heterochromatic DNA replication and genome stability by preferentially binding histone H3.1 over its close homolog, histone H3.3. The structure shows that TSK employs a shallow pocket in its poly-tetratricopeptide repeat (TPRTSK) domain to discriminate between these two variants. Structural analysis of TPRTSK bound to H3.1 revealed several additional binding pockets. Structural and functional assays revealed pockets that are important for histone H3.1 binding and TONSL-dependent DNA repair. Consistently, sponastrime dysplasia mutations mapping to the tetratricopeptide repeat of TSK-LIKE (TONSL) impairs TPRTSK binding to histone H3.1 and are unable to rescue the DNA repair function of TONSL in mammalian cells. Further characterization of the interaction between TPRTSK and N-terminal tail of H3.1 revealed that this interaction is highly regulated by post-translational modifications (PTMs) deposited on histone H3.1 during mitosis. Surprisingly, TPRTSK contains two novel pockets which cooperate to recognize monomethylated H3.1 K4 and acetylated H3.1 K27 (H3.1K4me1K27ac), two modifications that are associated with regulatory elements such as enhancers and promoters. Finally, our findings suggest that the TPRTSK must retain conformational flexibility to efficiently bind H3.1, as mutations that rigidify the protein impair this interaction. Overall, our findings revealed molecular interactions between histone H3.1 and the poly-TPR domain of TSK that are modulated in a multi-PTM-dependent manner and functionally relevant for the DNA repair functions of TONSL.

Dataset DOI: 10.5061/dryad.v41ns1sbw

Description of the data and file structure

Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

An HDX-MS dataset, characterizing the binding of peptide H3.1 to XlTONSL TPR1-461, is described. Briefly, TPRTONSL was equilibrated with a 4X molar excess of H3.1 peptide (12 μM:50 μM) and kept at 4 ⁰C for the entirety of the experiment. Deuterium labeling of the H3.1- TPRTONSL complex and TPRTONSL alone was initiated by a two-fold dilution with deuterated buffer (5 mM HEPES pH 7, 20 mM NaCl, 5 mM 2-mercaptoethanol, and 10 % glycerol in 80 % D2O, cooled to 4 ⁰C). Samples were labelled for 900 and 86 400 seconds and quenched by a ten-fold dilution with 200 mM TCEP, 2 M Urea in 0.1 % formic acid at 4 °C. Samples were manually injected and digested with an immobilized pepsin column (5 cm, prepared in house at 4 °C, followed by separation with on-line size exclusion chromatography (Acuity UPLC Protein BEH SEC Guard Column, 125 Å, 1.7 µm, 4.5 mm x 30 mm) at 4 °C at 100 µL/min for 310 seconds. Peptides were trapped (ACQUITY UPLC BEH C18 VanGuard Pre-column, 130 Å, 1.7 µm, 2.1 mm X 5 mm), separated (ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 100 µm x 100 mm) with a 10-35 % acetonitrile gradient over 6 minutes, and analyzed with an Orbitrap Eclipse (Thermo Fisher Scientific). A peptide map was generated with a database search (Mascot and MS Studio v.2.4.3667) unlabeled TPRTONSL sample collected in data-dependent acquisition mode. Deuteration was assigned and normalized to the theoretical maximum deuterium of each peptide (40 % D2O) with MS Studio v.2.4.3667. Differential HDX (Δ % D) was assessed by subtracting the deuterium content of the unbound from the protein-peptide complex (Δ % D = % D TONSL+ H3.1 – % D TONSL). Significance was assessed using a two-state Welch’s T-test with a 1-p threshold of 0.98 and a 3x standard deviation cut-off.

This dataset allows for insights into the unique deuterium uptake profile of TONSL when complexed with H3.1, and corroborates the structural findings from X-ray crystallographic experiments.

Files and variables

File: TONSL_HDXMS_Dataset.zip

Description:

The dataset includes the data-dependent mapping sample, and triplicate samples at two labelling timepoints for each of the protein states included in the differential HDX-MS experiment.

A full list of the data files and structure is described in the file named: HDX_MS_sample_list.xlsx.

The user can generate a peptide list from the undeuterated sample by searching against the include protein list (TONSL_protein_database.fasta). Otherwise, the user can import the included peptide list (TONSL_peptide_list_and_search_parameters.csv) into any HDX-MS software to interpret the raw data. For extracting differential HDX-MS data, the unbound TONSL samples should be used as the reference state, and the TONSL_H3.1 peptide as the bound samples. Differential deuteration is calculated by subtracting the deuteration of the unbound state from the bound state. The processed deuteration measurements (TONSL_MS_Studio_results.csv) results are included, should the user wish to directly inspect the processed output from MS Studio (the HDX-MS processing software used in this study).

The column headers in TONSL_MS_Studio_results.csv are as follows:

Protein state: Name of the sample
Start: Sequence number of the first amino acid in the peptide
Stop: Sequence number of the last amino acid in the peptide
Sequence: Amino acid sequence of the identified peptide
Charge: Peptide charge state
Peptide mass (Da): Monoisotopic peptide mass in Daltons
Retention time (min): Chromatographic retention time in minutes
HDX time (min): Experimental labelling time in minutes
Uptake (D): Mean absolute deuteration uptake measured in Daltons (triplicate)
Uptake SD (D): Standard deviation of the mean absolute deuteration uptake measured in Daltons (triplicate)
Uptake (%): Mean normalized deuteration uptake (triplicate)
Uptake SD (%): Standard deviation of the mean normalized deuteration uptake (triplicate)

Code/software

The .raw files (generated on an Orbitrap Eclipse (Thermo Fisher Scientific)) can be opened and viewed using proprietary platforms such as XCalibur (Thermo Fisher Scientific), MZMine, or alternative open-source platforms such as Skyline or OpenMS. These files can be imported into proprietary HDX-MS analysis platforms such as Mass Spec Studio (Trajan Scientific). Alternatively, deuterium can be calculated using open source software such as “The Deuterium Calculator” (https://pubs.acs.org/doi/10.1021/acs.jproteome.2c00558).