Role of ZRANB3 ATPase translocase in genome stability

The Zinc finger RAN-binding domain-containing protein 3 (ZRANB3) is a multifunctional protein encoded by the ZRANB3 gene in humans. It plays a crucial role in DNA repair and replication stress response, making it a significant target in diseases with impaired DNA repair mechanisms, such as cancer. ZRANB3 functions by processing DNA intermediates that arise during replication stress, and thus maintaining genome stability. Despite its importance, the structural basis of ZRANB3 and its action are yet unknown. The aim of this study was to conduct a preliminary analysis on ZRANB3 through the use of bioinformatic tools and experimental techniques, with the primary goal to produce a suitable amount of protein for structural studies, in particular X-ray crystallography and Cryo-Electron Microscopy (Cryo-EM). We performed sequence, sequence-structure and structure alignments using Clustal Omega, PROMALS3D and DALI light, respectively, and identified the putative ZRANB3 DNA binding residues. AlphaFold was utilized to predict the 3D structures of the full-length wild-type (FL WT) protein, which revealed key structural features. In particular, our attention focused on the C-terminal Substrate Recognition Domain (SRDct) and the ATPase motor domain (RecA-like 1/2), which are necessarily for the protein function. Indeed, the latter was used as substrate for docking a dsDNA molecule based on the information described above. Experimentally, the FL WT ZRANB3 gene along with SRDct and RECA1A2 domains were cloned into the pET28+ vector and initially tested for expression in small-scale in BL21 cells. Further, attempts to optimize the protein expression in large scale resulted in the formation of inclusion bodies and/or insoluble aggregates. Finally, we cloned the FL WT ZRANB3 in yeast P. pastoris aimed at Cryo-EM structural investigation. These preliminary experimental results serve as groundwork for optimization of the large-scale protein preparation for structural studies, which potentially can aid to the development of therapeutic strategies targeting cancer tissues.