Chan Siu-hong, Zhu Zhenyu, Van Etten James L, Xu Shuang-yong
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Nucleic Acids Res. 2004 Nov 29;32(21):6187-99. doi: 10.1093/nar/gkh958. Print 2004.
The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.
本文描述了小球藻病毒 NYs-1 编码的 CviPII DNA 切口和修饰系统的克隆与表达。该系统由一个共线性的甲基转移酶编码基因(cviPIIM)和一个切口内切核酸酶编码基因(cviPIINt)组成,二者被 12 个核苷酸隔开。M.CviPII 具有 C5 甲基转移酶典型的八个保守氨基酸基序(I 至 VIII),但与另一种小球藻病毒甲基转移酶 M.CviJI 一样,缺少保守基序 IX 和 X。除了对 CCD(D = A、G 或 T)序列中的第一个胞嘧啶进行修饰外,M.CviPII 还能对 CCAA 和 CCCG 位点的前两个胞嘧啶进行修饰。Nt.CviPII 与识别重叠序列(RG--CY)的 II 型限制性内切核酸酶 CviJI 具有显著的氨基酸序列相似性。Nt.CviPII 在经 M.CviPII 预修饰的宿主中,有或没有 His 标签的情况下在大肠杆菌中表达。重组 Nt.CviPII 识别 DNA 序列 CCD,并切割第一个胞嘧啶 5' 端的磷酸二酯键,而该位点的 DNA 另一条链不受影响。Nt.CviPII 表现出对 CCR(R = A 或 G)位点的偏好,CCR 位点优于 CCT 位点。Nt.CviPII 在 16 至 65 摄氏度下具有活性,最适温度为 30 - 45 摄氏度。Nt.CviPII 可用于生成单链 DNA(ssDNA)用于等温链置换扩增。Nt.CviPII 与 Bst DNA 聚合酶 I 大片段联合使用,可从基因组 DNA 或单个细菌菌落中快速扩增匿名 DNA。