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

摘要

PKZ was the most recently discovered member of eIF2 alpha kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2a kinase catalytic domain in C-terminal and two Z-DNA binding domains (Z alpha) in N-terminal. The Z alpha 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-Z alpha): P-Z alpha 1Z alpha Z2, P-Z alpha 1Z alpha 1 and P-Z alpha 2Z alpha 2. 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-Z alpha to the recombinant plasmids. Meanwhile, a competition experiment using P-Z alpha 1Z alpha Z2 and anti-Z-DNA antibody was performed. The results revealed that P-Z alpha 1Z alpha Z2 and P-Z alpha 1Z alpha Z2 were able to bind to the recombinant plasmids with high affinity, whereas P-Z alpha 2Z alpha 2 could not bind to it. In addition, dimerization of Patizaz indicated the function unit of Z alpha of CaPKZ would be a dimer. (C) 2010 Elsevier Ltd. All rights reserved.