Biochemical characterization of the PRDM9 Zinc-Finger array binding and investigating of multimer formation
Sprache des Vortragstitels:
FEBS Advanced Course Ligand-Binding Theory and Practice
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PRDM9 (PR-domain containing 9) has been identified as a meiosis specific protein that plays a major role in determining the location of meiotic recombination hotspots, but so far the function of PRDM9 has still been enigmatic. PRDM9 is an epigenetic modifier which binds DNA via its long zinc-finger (ZnF) array and directs double strand breaks necessary for the initiation of recombination in its close vicinity. Motifs recognized by the ZnF array of PRDM9 are enriched at the center of the hotspots, yet these motifs are neither necessary nor sufficient to determine the binding, and it is still unclear what factors drive the binding affinity and specificity of the ZnF array in-vivo.
For this purpose we characterized the binding specificity of PRDM9 (murine CAST allele with 11 zinc-fingers) to a target recombination hotspot, Hlx1, in-vitro, using Electrophoretic Mobility Shift Assays. By consecutively replacing the specific target site by 5-nucleotide steps with a random DNA sequence, we observed that a minimal number of 15 nucleotides confer binding specificity, located in the middle or at the 5? end of the sequence, whereas, 15 nucleotides at the 3? end still confer binding, but with a much lower affinity. This is consistent with binding assays assessing the effect of single nucleotide changes and with the observation that motif enrichments at hotspots are much shorter than the minimal binding site, fitting the paradoxical observation that PRDM9 is highly specific and permissive at the same time. In order to assess the binding affinity of PRDM9 to its specific target DNA we used gel shift assays as well as a more quantitative method, the switchSENSE technology from Dynamic Biosensors, to measure binding on and off kinetics resulting in a specific dissociation constant in the nM range. Furthermore we demonstrated that PRDM9 forms functional multimeric complexes of at least two or more monomer units that are mediated within the C-terminal Zinc-Finger domain.