Characterizing the Zinc Finger Binding Interactions of PRDM9 with the DNA of Recombination Hotspots
Sprache des Vortragstitels:
FEBS-EMBO 2014 Conference
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Meiotic recombination events cluster in 1-2 kb regions of the genome termed recombination hotspots. Hotspots have been characterized for a long time, but little is known on how and why a region is targeted for recombination. The histone methyltransferase PRDM9 has been identified as a key trans-regulator of hotspot activity in mammals. It is believed that PRDM9 recognizes specific DNA motifs via its tandem array of zinc fingers and then epigenetically marks the local chromatin by its histone methyltransferase activity. Specific DNA motifs recognized by PRDM9 are a key factor in determining hotspots, yet there are many cases in which motifs are neither necessary nor sufficient to determine a hotspot, and in many instances the motifs are found more often outside than within hotspots. Thus, we still do not fully understand the binding determinants of PRDM9 to DNA. We are therefore analyzing PRDM9 binding affinities to diverse DNA substrates in-vitro using Electrophoretic Mobility Shift Assays (EMSAs), as well as more quantitative biophysical techniques. We have confirmed that binding to a specific hotspot sequence is much stronger than to a random DNA sequence or even to the predicted binding sequence (derived by the Persikov algorithms). Moreover, the addition of high amounts of specific or unspecific DNA leads to an increased PRDM9 affinity, suggesting either a cooperativity effect or a change in accessibility to the DNA-binding region. Furthermore, epigenetic modifications such as DNA methylation as well as the nature of the regions flanking the motif have a strong effect on the binding. Our results show that DNA binding motifs recognized by the zinc finger array do not capture all the aspects of the binding site information.