Project start: January 2015

Recombination hotspots are very common throughout the genome, but it is still a mystery how these hotspots are controlled and what drives their activity. A few years ago, several groups identified PRDM9 as a key player in hotspot activity, but so far the function of PRDM9 is still enigmatic. PRDM9 is an epigenetic modifier which binds DNA via its Zn fingers and targets double strand breaks necessary for the initiation of recombination in its vicinity. 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. We clearly do not fully understand the binding determinants of PRDM9 to DNA, and sequence motifs do not capture all the aspects of the binding site information. With more information about binding characteristics of PRDM9 and the biochemical characterization of PRDM9 binding, some of the incongruous and bizarre observations about PRDM9 could be better addressed.

The aim of this proposal is to characterize PRDM9 binding in several human hotspots with and without the Myers motif. Specifically, we will analyze 1) the binding kinetics of human PRDM9 (variant A) to sequences of representative human hotspots; 2) the factors that influence the binding affinity and specificity, and 3) if reduced fertility is associated with allelic differences in PRDM9. In order to address these aims, we propose to characterize hotspots using sperm typing techniques and combine standard in vitro binding methods such as electrophoretic mobility shift assays (EMSA) with microscale thermophoresis (MST), a highly quantitative biophysical approach that can accurately measure binding affinities (dissociation constant; K_D) in unpurified substrates. With this project we can elucidate for the first time the components of PRDM9 binding and what features influence PRDM9 specificity and determine the regions targeted for meiotic recombination.