Biochemical characterization of the PRDM9 Zinc-Finger array binding

Description

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. For this purpose, we characterized biochemically the binding determinants of the PRDM9 ZnF array (murine CAST allele with 11 zinc-fingers). Specifically, we tested different sizes, arrangements, and sequence modifiers of the Hlx1 sequence and assessed the binding to the ZnF array using Electrophoretic Mobility Shift Assays (EMSAs), compatible with crude protein lysates. Given that every finger of the C2H2-type ZnF array binds three nucleotides, and we observed that 31- bind equally well as 34-nucleotide targets, it seems that all zinc-fingers of the array are crucial in the binding, except for the first degenerate C2H2-type ZnF. Interestingly, extending the available DNA sequence by 4 more nucleotides enhances the binding even further, which suggests that more nucleotides are required than number of zinc-fingers. It is likely that these additional nucleotides are necessary for a sliding-type recognition of the ZnF array, although DNA accessibility and rigidity might also play a role. We also assessed which zinc-fingers confer binding specificity by consecutively replacing the specific binding site by 5-nucleotide steps with a random DNA sequence...

Period	31 Aug 2015
Event title	EMBO Meiosis Meeting 2015
Event type	Conference
Location	United KingdomShow on map