The Interaction Between The Zα Bomain Of Fish PKZ (Protein Kinase Containing Z-DNA Binding Domain) And The Z-DNA Conformer In Negative Supercoils

Eukaryotic initiation factor 2a (eIF2a) kinases, a member of the highly conserved Ser/Thr protein kinases family, are distributed in different mammalian tissues and cells and mainly participated in a variety of emergency responses by regulating the activity of eIF2a. The eIF2a kinases mostly contain HRI, PKR, PERK and GCN2. PKZ, including CaPKZ, DrPKZ, AsPKZ, and GrPKZ, is the most recently discovered member of eIF2a kinase family in fish. PKZ has a special structure, which possessed a conserved eIF2a kinase catalytic domain in C-terminal and two Z-DNA binding domains (Za) in N-terminal. Therefore, PKZ belongs to the Za protein family together with ADAR1, DLM-1 and E3L.Z-DNA, different from B-DNA, adopted unique conformation and to be thougth to have many important biological functions. In order to mimic the Z-DNA under physiological conditions in vitro, we constructed the recombinant plasmids of pMD-18T/d(GC)n (n=6,8,10,13) that were detected by using inhibition of methylation experiments and anti-Z-DNA antibody. The results showed that most of the plasmids containing d(GC)n inserts were maintained in the Z-conformation. Moreover, the ability of the recombinant plasmid to form Z-DNA depended on the number of d(GC) repeats. It suggested that the longer the sequence of d(GC)n the more efficient it can form the Z-conformation. Meanwhile, we have also constructed the recombinant plasmids of MD-18T/d(TA)n and pMD-18T/d(non-GC-repeat)n that were detected by using anti-Z-DNA antibody. The results showed that both of them couldn't bind to the anti-Z-DNA antibody.CaPKZ is the first identified and reported PKZ in fish. To further investigate the function of CaPKZ Zα, we constructed three recombinant expression vectors of pET-22b(+)/Zα1Zα2, pET-22b(+)/Zα1Zα1 and pET-22b(+)/Zα2Zα2 by using pET-22b(+). Also, we constructed nine mutation vectors of pET-22b(+)/Zα(K34A, S35A, N38A, R39A,Y42A, K56A, P57A, P58A and W60A) by PCR site-directed mutagenesis method. These recombinant expression vectors were transformed into E.coli BL21 (DE3) plysS and then induced with IPTG. All kinds of purified peptides were obtained by using Ni-NTA His-Bind Resin affinity chromatography.The dimerization of PZα1Zα2, Pzα1Zα1 and PZα2Zα2 were analyzed by a 12%native polyacrylamide gel. The results showed that PZα1Zα2 could form dimer other than polymers in vitro. When incubated with 2-ME or SDS, the dimerization of PZα1Zα2 was inhibited. It suggested that disulfide bond could play a major role in stabilization of the dimer of PZα1Zα2.whereas pMD18-T/d(GC)6 had little effect on it. In contrast, no dimerization of PZα1Zα1 and PZα2Zα2 were detected.The recombinant plasmids of pMD18-T/d(GC)n, pMD-18T/d(TA)n and pMD-18T/d(non-GC-repeat)n binding activity of all kinds of fusion peptides were examined by using agarose gel mobility shift assay. At first, we found that both PZα1Zα2 and PZα1Zα1 had high affinity binding to the recombinant plasmids, and that of PZα1Zα1 could be stronger than of PZα1Zα2.If the negative supercoils of pMD18-T/d(GC)n were removed, the band shifts could disappeared. It suggested that the Z-conformation of pMD18-T/d(GC)n might be stabilized by negative supercoiling. At the same time, binding specificity of PZα1Zα2 to the plasmids of pMD18-T/d(GC)n was also examined by a competitive binding assay using the anti-Z-DNA antibody. On the other hand, PZα2Zα2 and nine site-directed mutation PZαcould not bind to pMD18-T/d(GC)n that lost its band shifting activity.In addition, we examined the binding activity of PZα1Zα2 to the recombinant plasmids of pMD-18T/d(TA)n and pMD-18T/d(non-GC-repeat)n by using agarose gel mobility shift assay. The results indicated that PZα1Zα2 has lower affinity binding to them, especially weakest to pMD-18T/d(TA)n.Additionally, the full length cDNA of grass carp(Ctenopharyngodon idellus) CiPKZ and its promoter sequence were cloned. Sequence analysis showed that grass carp CiPKZ shared the highest homology with crucian carp CaPKZ. The RT-PCR analysis showed that CiPKZ had a low level of constitutive expression in spleen, kidney and liver, and it was significantly up-regulated after challenged by Poly I:C. These results showed that the fish PKZ could be induced by virus, and and involved in host defense and immune response.