title: The Zα domain of fish PKZ facilitates the B-Z conformational transition of oligonucleotide DNAs with d(GC)(n) inserts.
abstract: PKZ (PKR-like) was discovered as a member of elF2α kinase family in fish, which possesses a conserved catalytic domain of an eIF2α kinase in C-terminal and also two Z-DNA-binding domains (Zα1 and Zα2) in N-terminal. PKZ can be activated through binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα still remains unclear. To investigate a molecular mechanism of how PKZ Zα interacts with Z-DNA, we expressed Zα polypeptide Zα1α2 in Escherichia coli Rosetta strain and purified by affinity chromatography on Ni-NTA resin. Different lengths of oligonucleotide DNAs with various inserts, namely d(GC)(n) (n = 6, 8, 10, 13), d(TA)(n) (n = 6, 10), non-d(GC), and non-d(TA), were designed and synthesized. Circular dichroism spectrum and gel mobility shift assays were used to investigate the effects of Zα1α2 on the conformational transition of different oligonucleotide DNAs. Results showed that oligonucleotide DNAs retained a conventional B-DNA conformation in the absence of Zα1α2. With the increasing amount of Zα1α2 titration, d(GC)(n) were recognized and converted to Z-DNA conformation to some degree. With increasing the repeat number (from n = 6 to n = 13), the tendency of conformational transition became more obvious. However, the conformation of oligonucleotides with d(TA)(n) inserts changed a little in the presence of Zα1α2, and Zα1α2 had no effect on conformational transition of oligonucleotides with non-d(GC) or non-d(TA) inserts. Gel mobility shift assays further showed that Zα1α2 could bind to oligonucleotide with d(GC)(10). In other words, Zα1α2 can turn oligonucleotides with d(GC)(n) inserts into Z-DNA conformation and bind to it with high affinity.
authors.affiliation: Department of Bioscience,  College of Life Science and Food Engineering,  Nanchang University, Nanchang 330031,  China.
authors.forename: Puzhong
authors.initials: P
authors.lastname: Lu
authors.forename: Shoulong
authors.initials: S
authors.lastname: Deng
authors.forename: Youlin
authors.initials: Y
authors.lastname: Zhu
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Yan
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Chengyu
authors.initials: C
authors.lastname: Hu
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D005399:Fishes; D009690:Nucleic Acid Conformation; D011487:Protein Conformation
chemical_list: D004247:DNA