New England Biolabs, Inc, Research Department, 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Res. 2013 Jan 7;41(1):378-90. doi: 10.1093/nar/gks1043. Epub 2012 Nov 3.
Many bacteriophage and prophage genomes encode an HNH endonuclease (HNHE) next to their cohesive end site and terminase genes. The HNH catalytic domain contains the conserved catalytic residues His-Asn-His and a zinc-binding site CxxC. An additional zinc ribbon (ZR) domain with one to two zinc-binding sites ([CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC] or [CxxH]) is frequently found at the N-terminus or C-terminus of the HNHE or a ZR domain protein (ZRP) located adjacent to the HNHE. We expressed and purified 10 such HNHEs and characterized their cleavage sites. These HNHEs are site-specific and strand-specific nicking endonucleases (NEase or nickase) with 3- to 7-bp specificities. A minimal HNH nicking domain of 76 amino acid residues was identified from Bacillus phage γ HNHE and subsequently fused to a zinc finger protein to generate a chimeric NEase with a new specificity (12-13 bp). The identification of a large pool of previously unknown natural NEases and engineered NEases provides more 'tools' for DNA manipulation and molecular diagnostics. The small modular HNH nicking domain can be used to generate rare NEases applicable to targeted genome editing. In addition, the engineered ZF nickase is useful for evaluation of off-target sites in vitro before performing cell-based gene modification.
许多噬菌体和原噬菌体基因组在其粘性末端位点和终止酶基因旁边编码一个 HNH 内切核酸酶 (HNHE)。HNH 催化结构域包含保守的催化残基 His-Asn-His 和一个锌结合位点 CxxC。在 HNHE 或位于其附近的 ZR 结构域蛋白 (ZRP) 的 N 端或 C 端，通常会发现一个额外的锌指 (ZR) 结构域，其中包含一个到两个锌结合位点 ([CxxxxC]、[CxxxxH]、[CxxxC]、[HxxxH]、[CxxC] 或 [CxxH])。我们表达并纯化了 10 种这样的 HNHE，并对其切割位点进行了表征。这些 HNHE 是具有 3-7bp 特异性的位点特异性和链特异性的切口内切核酸酶 (NEase 或尼克酶)。从芽孢杆菌噬菌体 γ HNHE 中鉴定出一个 76 个氨基酸残基的最小 HNH 切口结构域，并随后将其融合到锌指蛋白上，生成具有新特异性 (12-13bp) 的嵌合 NEase。大量以前未知的天然 NEase 和工程化 NEase 的鉴定为 DNA 操作和分子诊断提供了更多的“工具”。小而模块化的 HNH 切口结构域可用于生成适用于靶向基因组编辑的稀有 NEase。此外，工程化的 ZF 尼克酶可用于在进行基于细胞的基因修饰之前，在体外评估脱靶位点。
