Journal: bioRxiv
Article Title: Hydra domain drives SNF2L multimerization and marks ISWI diversification in parasites
doi: 10.1101/2025.09.03.673926
Figure Lengend Snippet: a . Recombinant Tg SNF2L and its hydra domain deletion variant (Δhydra) were purified and analyzed by 4-12% NuPAGE, followed by Coomassie blue staining and anti-His tag Western blotting. b . Nucleosome remodeling assay using restriction enzyme accessibility confirms that both full-length and Δhydra recombinant Tg SNF2L retain catalytic activity. Commercial Hs SNF2h (top), recombinant full-length Tg SNF2L (middle), and truncated Tg SNF2L lacking the Hydra domain (bottom) were incubated with EpiDyne nucleosome remodeling substrates. In this assay, remodeling exposes previously occluded GATC sites, enabling cleavage by the restriction enzyme DpnII. The upper band corresponds to intact nucleosomes; the appearance of the lower band indicates successful remodeling. The first lane serves as a -DpnII control, subsequent lanes represent increasing reaction times and the final lane is - ATP control. c . Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALLS) shows that removing the hydra domain decreases the higher oligomeric forms of Tg SNF2L in the micromolar range. With the loss of the hydra domain, two new forms are detected, corresponding to a Tg SNF2L and Tg SNF2LΔhydra SEC-MALLS (Superose 6 Increase) chromatograms shown as the refractive index curves in blue and orange, respectively. Point measurements of the molecular weight in kDa are displayed as black curves with average masses within the peak regions. d . Mass photometry demonstrates a decrease in tetramer and higher oligomeric forms in the nanomolar range upon hydra domain deletion. The data, shown as normalized counts per molecular weight bin (one representative experiment), compares Tg SNF2L and Tg SNF2LΔhydra in blue and orange, respectively. Monomer, dimer and tetramer peaks are fitted using Gaussian distribution model while higher oligomeric forms are delimited by a dotted line. The relative quantifications of these peaks or windows are shown on the right with the mean and standard deviations shown from duplicate measurements. e . Proposed model: The hydra domain acts as a multimerization module, facilitating Tg SNF2L storage in a functionally primed state. In this model, Tg SNF2L’s multi-oligomeric forms may rapidly release Tg SNF2L and its associated proteins in response to DNA damage or replication fork progression.
Article Snippet: SNF2L Hydra domain (886-1057) was codon optimized for E. coli, synthetized and cloned by Genscript within a modified pET30-a (+) vector (Addgene) in order to possess a C Terminal, TEV cleavable, dual 6*His Tag.
Techniques: Recombinant, Variant Assay, Purification, Staining, Western Blot, Activity Assay, Incubation, Control, Size-exclusion Chromatography, Multi-Angle Light Scattering, Refractive Index, Molecular Weight
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