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

PKZ was the most recently discovered member of eIF2alpha kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2alpha kinase catalytic domain in C-terminal and two Z-DNA binding domains (Zalpha) in N-terminal. The Zalpha 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 (P(Zalpha)): P(Zalpha1Zalpha2), P(Zalpha1Zalpha1) and P(Zalpha2)(Zalpha2). 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 P(Zalpha) to the recombinant plasmids. Meanwhile, a competition experiment using P(Zalpha1Zalpha2) and anti-Z-DNA antibody was performed. The results revealed that P(Zalpha1Zalpha2) and P(Zalpha1Zalpha1) were able to bind to the recombinant plasmids with high affinity, whereas P(Zalpha2)(Zalpha2) could not bind to it. In addition, dimerization of P(Zalpha1Zalpha2) indicated the function unit of Zalpha of CaPKZ would be a dimer.