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Crystal structure of Cas1 in complex with branched dna

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Science China(Life Sciences) 2020年第4期63卷 516-528页

作者： Jing Yang Jiazhi Li Jiuyu Wang Gang Sheng Min Wang Hongtu Zhao Yanhua Yang Yanli Wang Institute of Life Sciences Jiangsu UniversityZhenjiang 212013China Key Laboratory of RNA Biology CAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China National Laboratory of Biomacromolecules CAS Center for Excellence in BiomacromoleculesInstitute of BiophysicsChinese Academy of SciencesBeijing 100101China

Cas1 is a key component of the CRISPR adaptation complex,which captures and integrates foreign dna into the CRISPR array,resulting in the generation of new *** have determined crystal structures of Thermus thermophilus Cas1 involved in new spacer acquisition both in complex with branched dna and in the free ***1 forms an asymmetric dimer without ***,two asymmetrical dimers bound to two branched dnas result in the formation of a dna-mediated tetramer,dimer of structurally asymmetrical dimers,in which the two subunits markedly present different *** the dna binding complex,the N-terminal domain adopts different orientations with respect to the C-terminal domain in the two monomers that form the *** binding triggers a conformational change in the loop 164–177 *** loop is also involved in the 3′fork arm and 5′fork arm strand recognition in monomer A and B,*** study provides important insights into the molecular mechanism of new spacer adaptation.

关键词： CRISPR Cas protein Cas1 adaptation dna integration structure protein-dna complex

The applications of nanopores in studies of proteins

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Science Bulletin 2019年第19期64卷 1456-1467页

作者： Taoli Ding Antony KChen Zuhong Lu Department of Biomedical Engineering College of EngineeringPeking UniversityBeijing100871China State Key Laboratory of Bioelectronics School of Biological Science and Medical EngineeringSoutheast UniversityNanjing210096China

Nanopores are a label-free platform with the ability to detect subtle changes in the activities of individual biomolecules under physiological ***,we comprehensively review the technological development of nanopores,focusing on their applications in studying the physicochemical properties and dynamic conformations of peptides,individual proteins,protein-protein complexes and protein-dna *** is followed by a brief discussion of the potential challenges that need to be overcome before the technology can be widely accepted by the scientific *** believe that with continued refinement of the technology,significant understanding can be gained to help clarify the role of protein activities in the regulation of cellular physiology and pathogenesis.

关键词： Nanopores proteins protein-protein interactions protein-dna interactions

Interaction of zinc finger ZNF191(243—368) and its I323W and R327W mutants with oligonucleotides by florescence spectrum

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Science China Chemistry 2003年第1期46卷 64-74页

作者：周晶刘玉奇汤李明滕昕辰唐宁黄仲贤 Department of Chemistry Fudan University Shanghai 200433 China School of Chemistry and Chemical Engineering Lanzhou University Lanzhou 730000 China

Two mutants of the zinc finger protein, ZNF191(243—368) I323W and R327W, are suc-cessfully obtained by site-directed mutagenesis. The fluorescence spectrum is used to study the interaction between these two mutants and the oligonucleotides. The influence of the mutation on the interaction has been studied using ethidium bromide (EB) as the fluorescence probe. The binding constants of the I323W-dna and R327W-dna have been calculated and the possible binding models have been discussed.

关键词： zinc finger protein ZNF191(243-368), site-directed mutagenesis, fluorescence spectrum, protein-dna interaction.

Evolutionary conservation and dna binding properties of the Ssh7 proteins from Sulfolobus shibatae

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Science China(Life Sciences) 2002年第6期45卷 583-592页

作者：陈绪林郭荣黄力 Ray Hong Pomona College CA91711

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the c/s-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex dna fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with dna is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex dna fragments with a binding size of - 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled dna than to linear or relaxed dna.

关键词： hyperthermophilic archaea, dna binding proteins, ssh7a/ssh7b genes, protein-dna interaction.