Exploring the DNA-binding specificities and kinetics of the zinc finger protein PRDM9
Sprache des Vortragstitels:
Englisch
Original Tagungtitel:
EMBO Meiosis Meeting 2015
Sprache des Tagungstitel:
Englisch
Original Kurzfassung:
PRDM9 is a multi-domain protein capable of regulating the activity of recombination hotspots in mammals. It is believed that PRDM9 specifically recognizes DNA motifs via its tandem array of zinc fingers (ZnFs) and then epigenetically marks the local chromatin by its histone methyltransferase activity. The DNA motifs recognized by PRDM9 are a key factor in determining hotspot locations, yet these motifs are much shorter than the predicted DNA target sequence of the ZnF-array. DNA motifs are also abundant outside hotspots, and paradoxically ZnF-binding is highly specific, but also permissive with the recognition of alternative DNA sequences. Thus, it is still enigmatic how PRDM9 chooses its target DNA.
We therefore analyzed the binding properties and kinetics of PRDM9 to known hotspot DNA sequences in-vitro. Using a highly innovative technology, known as switchSENSE, we were able to gain first insights into the binding kinetics of the murine PRDM9cst ZnF-array to Hlx1-hotspot DNA. We observed that the ZnF-domain of PRDM9 exhibits an exceptionally slow association rate to Hlx1 DNA, but an even slower dissociation rate, resulting in a dissociation constant (Kd) in the nM range.
We also analyzed which ZnF-domains engage in DNA binding. As opposed to prevalent models suggesting that multi-zinc finger proteins contact DNA in units of 2-3 successive ZnFs, our results clearly show that 3 zinc fingers are not sufficient to confer binding specificity. We observed that a minimal number of 15 nucleotides, contacting 5 consecutive ZnFs, are required to establish a specific PRDM9-Hlx1 interaction. Moreover, our results indicate that on the level of DNA, deletions of specific sequences have a stronger effect on PRDM9 binding at the 5? than at the 3?-end, suggesting that the N-terminal ZnFs, exhibit a higher affinity. However, also the ZnFs at the C-terminal end can bind specifically to Hlx1, albeit with lower affinity.
Forschungs-