Journal: bioRxiv

Article Title: Reconstitution of SPO11-dependent double-strand break formation

doi: 10.1101/2024.11.20.624382

Figure Lengend Snippet: a , Domain organization of dimeric SPO11–TOP6BL complexes and Topo VI holoenzyme. Left, side view. Right, top views (with and without DNA) looking down into the DNA-binding channel. Catalytic tyrosine (Y), metal binding pocket, and hybrid active site (dashed circle) are shown. Reproduced from ref. under a CC-BY 4.0 license. b , Coomassie-stained SDS-PAGE of purified Flag SPO11–TOP6BL preparations (0.5 µg each). WT, wild type. c , Monomeric SPO11–TOP6BL complexes. Mass photometry profiles are shown without or with 5 mM ATP. Protein concentration was 28 nM. Particle counts (gray bars), gaussian density fits (red lines), fitted mean ± s.d., and percentages of total particles are shown. Asterisks, background material also present in blanks. d , EMSA of binding to DNA ends. SPO11 complexes were titrated with a 5′-labeled 25-bp hairpin substrate with a two-nucleotide 5′ overhang end. Quantification (mean ± s.d. of n = 3 experiments; apparent K d given as mean ± s.e.) is shown below for wild type and Y138F (gel image in Extended Data Fig. 1c ). e , DNA length dependence for double-end binding. Selected lanes from gel shift assays show binding of SPO11 complexes to DNAs of the indicated lengths with two-nucleotide 5′ overhangs on both ends (full gels in Extended Data Fig. 1d ). Quantification of double-end binding is shown below, with yeast Spo11 data for comparison. f , AFM analysis of binding to linearized plasmid DNA. Examples are shown of binding to ends (one-end), internally on duplex DNA (duplex), junctions of three DNA arms (three-way), and junctions of four DNA arms (four-way). Percentages are from n = 200 particles scored. g , Histogram of bending angles (n = 200 particles). Examples are from subpopulations with modal values of ∼60° and ∼120°, similar to yeast . Angles at randomly chosen positions along the DNA are shown as a control (n = 138 positions).

Article Snippet: The products were purified using the QIAquick PCR purification kit (QIAGEN), mixed with 6× DNA loading dye (NEB), and separated by agarose gel electrophoresis as in the DNA cleavage assays.

Techniques: Binding Assay, Staining, SDS Page, Purification, Protein Concentration, Labeling, Gel Shift, Comparison, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Reconstitution of SPO11-dependent double-strand break formation

doi: 10.1101/2024.11.20.624382

Figure Lengend Snippet: a , DNA cleavage assays with SPO11–TOP6BL complexes containing wild-type or Y138F SPO11. Reactions contained 4 ng/µl pUC19 DNA, 100 nM SPO11 complexes, and 5 mM MnCl 2 . Deproteinized samples were separated on agarose gels stained with SYBR Gold. A representative gel is shown above, quantification (mean ± s.d. of n = 3 experiments) is below. b-d , Covalent attachment of SPO11 to cleaved DNA. Panel b shows a schematic overview of experiments in c and d . Wild-type or Y138F SPO11 complexes (325 nM) were mixed on ice with 4 ng/µl DNA and 5 mM MnCl 2 in 60 µl and either immediately quenched with 0.5 % SDS (– reaction) or incubated at 37 °C for 11 min before quenching (+ reaction). In c , mixtures were centrifuged through CsCl cushions and the precipitated material was immuno-slot-blotted with anti-Flag antibodies (two exposure levels shown). No-protein negative controls (DNA only and buffer only) and positive controls for protein detection (10 ng) were included. In d , mixtures were immunoprecipitated with anti-Flag antibodies, then samples digested with proteinase K were separated by agarose gel electrophoresis. e , Differential prediction of 5′ covalent protein association with radiolabeled strands (asterisks) for 5′ vs. 3′ end-labeled substrates. f , Covalent attachment of SPO11 to 5′ ends. A 583 bp restriction fragment from pUC19 was either 5′ or 3′ radiolabeled on one end, then incubated with SPO11 complexes and separated by denaturing PAGE with or without prior digestion with proteinase K. g , Model to explain biphasic reaction kinetics. See text for details. h , Substrate order of addition determines reaction rate. SPO11 complexes (100 nM) were incubated with 4 ng/µl each of pUC19 (P1, 2.7 kb) and pCD-NA3.1-based plasmid mp134 (P2, 6.8 kb). Protein was mixed with both plasmids on ice before initiating reactions by transfer to 37 °C (simultaneous), or protein was incubated with one plasmid on ice and the second plasmid was added immediately before transfer to 37 °C (P1®P2 and P2®P1). Aliquots at the indicated times were quenched with SDS and deproteinated before agarose gel electrophoresis. Each plasmid (either supercoiled (SC) or linearized (LN)) was also run separately as a size marker. Representative gels are shown above, quantification (mean ± s.d. of n = 3 experiments) is below. Asterisk, slower migrating species that are likely to be multimers and/or catenated copies of P2.

Article Snippet: The products were purified using the QIAquick PCR purification kit (QIAGEN), mixed with 6× DNA loading dye (NEB), and separated by agarose gel electrophoresis as in the DNA cleavage assays.

Techniques: Staining, Incubation, Immunoprecipitation, Agarose Gel Electrophoresis, Labeling, Plasmid Preparation, Marker