Molecular weight of protein-DNA complex can be inferred from native gel electrophoresis

Description

Introduction: PRDM9 is a multi-domain protein responsible to determine the locations of meiotic recombination hotspots by recognizing specific DNA motifs via its repetitive array of zinc fingers (ZnFs). Recently it was shown that PRDM9 forms an active multimer; however, neither the size nor the factors inducing the multimerization are known. Materials and Methods: We performed in vitro binding studies, based on gel shift assays, to infer the size of the protein multimer using DNA fragments that increased in length, containing either one or two specific target sites. We tested different protein constructs including truncated protein versions, and analyzed the molecular weight and the corresponding migration distance of binding complexes in relationship to known standards to further determine the protein stoichiometry. Results: We observed that PRDM9 multimerizes to an active trimer which is formed within the repetitive DNA-binding ZnF domain and 5 out of 11 ZnF repeats are sufficient to form the trimer. Moreover, we demonstrated that only one of the ZnF arrays within the trimer contacts the DNA, whereas the remaining two ZnFs likely perform ZnF-ZnF interaction to maintain the multimeric conformation. Conclusions: In this work, we report a method using native gel electrophoresis to infer the size, as well as, to investigate the molecular interaction of a multimeric protein with DNA. This simple method is suitable especially for proteins with complex structures or repetitive motifs as it is the case for many ZnF proteins.

Period	16 Jun 2019
Event title	European Human Genetics Conference 2019
Event type	Conference
Location	SwedenShow on map