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The Zα domain of fish PKZ converts DNA hairpin with d(GC)n inserts to Z-conformation
作者姓名:Puzhong Lu  Shoulong Deng  Chuxin Wu  Youlin Zhu  Yong Liu  Gang Lin  Yongbin Yan  Chengyu Hu
作者单位:[1]Department of Bioscience, College of Life Science and Food Engineering, N anchang University, Nanchang 330031, China [2]State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and I3iotechnology, Tsinghua University, Beijing 100084, China [3]Nanchang Teachers College, N anchang 330103, China
基金项目:This work was supported by grants from the National Natural Science Foundation of China (30560116, 30860218).
摘    要:PKZ, protein kinase containing Z-DNA domains, is a novel member of the vertebrate eIF2α kinase family. Containing a catalytic domain in C-terminus and two Z-DNA binding domains (Zαl and Zα2) in N-terminus, PKZ can be acti- vated through the binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα remains to be established. Here, to understand the impact of PKZ Zα on DNA con- formational transition, wild-type ZαdZα2 and 11 mutant proteins were expressed and purified. At the same time, several different lengths of DNA hairpins-d(GC)nT4(GC), (n = 2-6) and an RNA hairpin-r(GC)6T4(GC)6 were synthesized. The effects of ZαdZα2 and mutant proteins on the conformation of these synthetic DNA or RNA hairpins were investigated by using circular dichroism spectrum and gel mobility shift assays. The results showed that DNA hair- pins retained a conventional B-DNA conformation in the absence of ZαdZα2, while some of the DNA hairpins (n 〉 3) were converted to Z-conformation under Zαd Zα2 induction. The tendency was proportionally associated with the increas- ing amount of GC repeat. In comparison with ZodZα2, ZαdZαd rather than Zα2ZαL2 displayed a higher ability in converting d(GC)6T4(GC)6 from B- to Z-DNA. These results demonstrated that Zcd sub-domain played a more essential role in the process of B-Z conformational transition than Zα2 sub-domain did. Mutant proteins (K34A, N38A, R39A, Y42A, P57A, P58A, and W60A) could not convert d(GC)6T4(GC)6 into Z-DNA, whereas S35A or K56A retained some partial activities. Interestingly, ZαlZα2 was also able to induce r(GC)6T4(GC)6 RNA from A-conform- ation to Z-conformation under appropriate conditions.

关 键 词:Z-DNA  构象转变  GC  DNA结合结构域  突变体蛋白  催化结构域  DNA转化  蛋白激酶
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