Affinity Of The Zα Domain From Carassius Auratus PKR-like(PKZ) Binding To The Recombinant Plasmid Containing D(GC) Or D(TA) Insert

Zαdomain can recognise and specially bind to left-handed DNA(Z-DNA).The Carassius auratus PKR-like (CaPKR-like)(PKZ), identifided as the first fish protein kinase, contains Z-DNA binding domain, Za. In order to insight the CaPKR-like Zαmotif, the cDNAs encoded substitution P_(Zα1)2(Zα1Zα1) and site-mutations (P_Zα^K34A,P_Zα^S35A, P_Zα^R39A P_Zα^P57A) constructed by site-directed mutagenesis were linked into experssion vector and transformed into BL21(DE3) pLysS, then induced by IPTG The mutation fusion peptides were obtained by affinity chromatography as wel as wildtype P_Zα1Zα2.At the same time, three recombinant plasmids for mimic Z-DNA, containing d(GC)₆ or d(GC)₁₃ or d(TA)₁₃ insert respectively, were successfully constrcuted as well as hairpins (d(GC)₃T₄d(GC)₃,d(GC)₆T₄d(GC)₆) and oligodeoxynucleotides (d(GC)₆, d(GC)₁₃). The results of methylation inhibition demonstrated that d(GC)₆ and d(GC)₁₃ inserts were "latent" Z-DNA. Theses implied that CaPKR-like Za peptide could bind to the inserted fragments d(GC)₆ or d(GC)₁₃ within the recombinant plasmids then gave rise to the band shift effection.The wild type P_Zα1Zα2 and substitution P_Zα1Zα1 rather than 4 mutations (P_Zα^K34A, P_Zα^S35A, P_Zα^R39A P_Zα^P57A) were capable to bind to the three recombinant plasmids in vitro and the affinity enhanced in the present of increasing amounts of protein. In addition, compared with affinity of wild type P_Zα, substitution P_Zα1Zα1 was stronger. 4 mutations (P_Zα^K34A, P_Zα^S35A, P_Zα^R39A, P_Zα^P57A) had no ability to bind to d(GC) and d(TA) recombinant plasmids by the gel mobility shift assay, this result suggested that the 4 amino acids are important to Za binding to DNA.The affinity was also detected by the native-PAGE that wild type P_Zα1Zα2 and substitution P_Zα1Zα1 binding to hairpins and oligodeoxynucleotides respectively. These peptides were weakly capable to bind to hairpins and oligodeoxynucleotides. No difference was observed between wild type P_Zα1Zα2 and substitution P_Zα1Zα1 binding to this kinds of nucleis acids.