Biochemical Characterization of a dsDNA-Dependent Translocase Fused with dCas9

Abstract

Superfamily 2 (SF2) helicase comprises a large number of proteins, which are functionally diverse and are present across all domains of life. Unlike conventional helicases, which are mostly known to perform dsDNA/RNA unwinding, SF2 helicases translocate on dsDNA using energy from ATP hydrolysis and form the core of proteins participating in essential cellular functions, viz. DNA replication, repair, and recombination; transcription; chromatin remodeling, etc. Despite exhibiting a large diversity in domain organisation, they share a conserved catalytic ATPase domain and translocate along dsDNA/RNA without showing any sequence specificity. In this study, we have engineered a dsDNA-dependent translocase by fusing a minimal ATPase from the SF2 helicase with dCas9 and to target it to a specific region on DNA. To achieve the aforementioned aim, we overexpressed the fused protein in E. coli and purified it. Biochemical activities were validated using the NADH-coupled ATPase assay to check its ATP hydrolysis activity and the triplex displacement assay to assess translocase activity. Finally, Cryo-EM work was initiated to determine the structure of the fused protein.