The Zα domain of PKZ from Carassius auratus can bind to d(GC)n in negative supercoils

PKZ was the most recently discovered member of eIF2α kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2α kinase catalytic domain in C-terminal and two Z-DNA binding domains (Zα) in N-terminal. The Zα of CaPKZ closely resembled that of other Z-DNA binding proteins: ADAR1, DLM-1, and E3L. In order to understand more about the function of CaPKZ, we expressed and purified three constructed peptides of CaPKZ (PZα): PZα1Zα2, PZα1Zα1 and PZα2Zα2. Moreover, most of the plasmids containing d(GC)n inserts were maintained in the Z-conformation, as confirmed by using inhibition of methylation experiments and anti-Z-DNA antibody. Gel mobility shift assays were then used to examine the affinity of these PZα to the recombinant plasmids. Meanwhile, a competition experiment using PZα1Zα2 and anti-Z-DNA antibody was performed. The results revealed that PZα1Zα2 and PZα1Zα1 were able to bind to the recombinant plasmids with high affinity, whereas PZα2Zα2 could not bind to it. In addition, dimerization of PZα1Zα2 indicated the function unit of Zα of CaPKZ would be a dimer.