Identification of genetic interactors of Cdb4 by Yeast-2-Hybrid screening

Abstract

Background/Objectives: Curved DNA binding protein (Cdb4) has unknown functions but binds to curved DNA in vitro in the fission yeast Schizosaccharomyces pombe. Previous study identified Nup184 as its synthetic lethal interactor. This study aims to further identify other interacting proteins of Cdb4 with a view to determining its biological function. Materials and Methods: Cdb4-containing bait plasmids were constructed by cloning the ORF (Cdb4_FL) or domain fragments (Cdb4_Nt, Cdb4_BDCt, Cdb4_Ctand Cdb4_t) into pGBKT7 backbone. The bait plasmids were first individually transformed into the S. cerevisiae host strain (AH109) and ~1 x 10⁹ of competent, bait-harboring AH109 cells were co-transformed with AD/cDNA library. Single colonies obtained from the low stringency plate were replica plated on medium and high stringency media and positive colonies further confirmed on a medium stringency medium containing 3-AT.  cDNA fragments from prey plasmids were amplified by colony PCR, sequenced and the identity of interacting gene determined by a BLAST search. The function of the identified gene/protein was obtained from the pombe database (Pombase). Results and Conclusion: Of the five bait plasmids constructed, only Cdb4_t produced colonies on the high stringency confirmation. Eight proteins were found to interact with Cdb4_t and the interactions were sustained in the presence of 3-AT. The proteins were involved in diverse functional pathways including cell wall-related proteins (Psu1, Gto1), mitochondrial protein (Tdh1/Gpd3), transcription factors (Tmf1, Rep1), ubiquitin hydrolase (Ubp2), lipid metabolism (Plb1) and stress-related proteins (Pdr13). Cdb4 is potentially involved in several important processes in the cell. Its unique C-terminal region is responsible for interaction with proteins in vivo. Further genetic assays are required to confirm the biological significance of these identified interactions.