Biochemical Characterization of PRDM9 Binding

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; KD) 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.

During meiosis the maternal and paternal genetic material is exchanged in a highly regulated process known as meiotic recombination. Recombination is clustered in narrow hotspot regions in the genome. In many mammals, the protein PRDM9 plays a crucial role in determining the locations of recombination hotspots by recognizing specific DNA sequences. However, there are instances of hotspots without this specific sequence and these sequence motifs are found more often outside than within hotspots. Since many molecular aspects of PRDM9 binding are still unclear, we analyzed in this project the interaction of PRDM9 Zn finger domain with specific DNA recognition sequences. Specifically, we characterized PRDM9 binding at the molecular level using quantitative biophysical technologies. We analyzed 1) the binding affinity and kinetics of PRDM9 (murine variant) to representative sequences of a murine hotspot; 2) the factors that influence the binding affinity and specificity; and 3) the protein stoichiometry of PRDM9 when interacting with its target sequence. With our data, we demonstrated that PRDM9 forms a highly stable complex when binding to its specific DNA binding site and only dissociates after several hours. Moreover, PRDM9 can use subsets of its binding domain and interact specifically with a variety of different DNA sequences, which might explain the plasticity of the motif recognition by this protein. Finally, we demonstrated that PRDM9 interacts with DNA as a multimeric complex formed by three protein units. This work shows for the first time the high stability of the PRDM9-DNA interaction, which is a key molecular feature for PRDM9 to carry out its activity during the initiation of recombination. Moreover, our analysis of the molecular interactions clearly shows that the multiple PRDM9 units within the complex bind only with one site. These findings contribute to our understanding of the role of this protein in the regulation of hotspots.