Biochemical characterization of the PRDM9 zinc-finger array binding
Sprache des Vortragstitels:
EMBO Conference Meiosis 2015
Sprache des Tagungstitel:
PRDM9 has been identified as a meiosis specific protein that plays a major role in determining the location of meiotic recombination hotspots. 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. 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. 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. Furthermore, we observed that a level of 50% methylated DNA strongly reduces the binding.