PRDM9 (PR-domain containing protein 9) is a multi-domain protein, expressed in male and female germ cells at the beginning of meiosis. Using its long DNA-binding zinc finger domain, it controls the sites of the genetic exchange between maternal and paternal chromosomes, so called "recombination hotspots". This protein plays an important role for the protection of genome integrity, since it directs hotspots away from regions with gene expression.
This work presents first insights into the kinetics of the PRDM9-DNA interaction. Using state-of-the-art, biophysical techniques such as switchSENSE (Dynamic Biosensor GmbH, Munich) and gel-shift assays (EMSA), we observed that PRDM9 forms a highly stable complex with specific hotspot DNA lasting many hours. Such a long-lived interaction may be required for a successful translocation of the hotspot DNA to the “recombination initiation machinery” that targets the DNA for a double strand break necessary for the start of meiotic recombination. Furthermore, we analyzed the effect of single nucleotide polymorphisms in the DNA on the affinity of PRDM9.
Moreover, this thesis tested several cell systems for the expression of recombinant murine PRDM9Cst addressing their advantages and disadvantages. The large size of PRDM9 and the repetitive nature of its zinc finger domain poses difficulties to express this protein in multiple hosts, which is further complicated by its insolubility and degradation. Furthermore, several attempts of purifying PRDM9 led to a loss of function. Here we present an optimized protocol for PRDM9Cst lysate preparation, yielding a sufficient level of purity to be compatible with switchSENSE and EMSA, without the need for an affinity purification.