Assessment of PRDM9 Zinc Finger Binding with DNA of Recombination Hotspots
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6th ÖGMBT Annual Meeting Life Science meet Enterpreneurship
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In the past few years, it was shown that PRDM9 plays a major role determining the location of meiotic recombination sites, known as recombination hotspots. With its long array of tandem zinc fingers, PRDM9 recognizes specific DNA motif sequences in a recombination hotspot, which is then epigenetically modified by the histone methyltransferase activity of the PRDM9 PR/SET domain. However, it is still not clear what factors influence the binding of PRDM9 to DNA, since not all hotspots contain a PRDM9 motif nor a motif sequence in the genome results in a hotspot per se. Thus, we developed an in-vitro system to investigate the binding between the recombinantly expressed PRDM9 zinc finger domain of the mouse CAST/EiJ strain to a 75bp double-stranded DNA of the Hlx1 hotspot, known to bind PRDM9. With electrophoretic mobility shift assays (EMSAs), compatible with crude protein extracts, we showed sequence specific binding, which is enhanced with excess DNA. We also tested different sizes and positions of the motif within the Hlx1 sequence, to obtain new information about the PRDM9 minimal binding site. Furthermore, epigenetic modifications such as DNA methylation, as well as the nucleotide composition of regions flanking the motif have a strong effect on the binding. Finally, competition and titration experiments have provided semi-quantitative information about the binding affinity and kinetics of PRDM9. With the current system it is possible to further explore individual zinc fingers and nucleotides directly involved in the PRDM9-DNA interaction to elucidate the factors influencing PRDM9 binding.