Investigation of the PRDM9 zinc-finger multimer formation
Sprache des Vortragstitels:
Abcam Conference: Mechanisms of Recombination
Sprache des Tagungstitel:
PRDM9 has been identified as a meiosis specific protein that plays a key role in determining the location of meiotic recombination hotspots. It binds DNA via its long zinc-finger (ZnF) array, marks surrounding nucleosomes via its PR/SET domain and additionally binds to other proteins via its KRAB domain helping to direct the double strand break machinery in its close vicinity necessary for the initiation of recombination. Recently, it has been suggested that PRDM9 acts as a multimer in-vivo. However, to date the number of units in an active PRDM9 multimer, as well as, which domains are involved in the multimerization are not known. Using in-vitro binding studies, we characterized the protein stoichiometry within the DNA-protein complex of two different murine PRDM9 alleles, PRDM9Cst and PRDM9Dom2. For this purpose, we used native gel electrophoresis and EMSA to visualize the complex. First, we established that the molecular weight of a PRDM9-DNA complex can be inferred from a native polyacrylamide gel by using DNA sequences of increasing lengths as standards. Moreover, we also assessed which domains of PRDM9 drive the multimerization using constructs missing distinct parts of the PRDM9 full-length protein. We observe that both tested alleles form functional multimeric complexes of three monomer units which are mediated within the variable ZnF domain. We also show that only one of the ZnFs in the trimeric PRDM9 complex binds to DNA with one DNA molecule bound at a time. Finally, we analyzed the PRDM9-DNA binding complex with mass spectrometry and confirmed that the complex is solely formed by PRDM9 and DNA. Our results confirm the in-vivo observation of PRDM9 multimerization and suggest that within a complex only one ZnF array is actively binding to its recognition site. This is an important aspect in terms of dosage in hybrids with two Prdm9 alleles, and could explain the dominance of one Prdm9 allele over the other in a heterozygous PRDM9 complex.