Zhu Hui, Shuman Stewart
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
J Biol Chem. 2006 May 19;281(20):13873-81. doi: 10.1074/jbc.M600055200. Epub 2006 Mar 14.
DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.
DNA连接酶D（LigD）在细菌的非同源末端连接过程中执行末端重塑和末端封闭反应。铜绿假单胞菌LigD由一个中央ATP依赖性连接酶结构域与一个C端聚合酶结构域和一个N端磷酸酯酶（PE）模块融合而成。PE结构域在引物模板的引物链3'端催化依赖锰的磷酸二酯酶和磷酸单酯酶反应。磷酸二酯酶切割3'末端的二核糖核苷酸，产生具有核糖核苷3'-PO4末端的引物链。磷酸单酯酶将引物模板的末端核糖核苷3'-PO4或脱氧核糖核苷3'-PO4转化为3'-OH。在此，我们报告磷酸二酯酶和磷酸单酯酶活性均依赖于引物模板底物5'单链尾巴的存在和长度。尽管磷酸二酯酶活性严格依赖于倒数第二个核糖的2'-OH，但对末端核苷上的2'-OH与2'-H不敏感。当2'-OH在5'方向上移动1个核苷酸时，核糖核苷酸连接处的切割受到抑制，这表明LigD是一种切割3'末端磷酸二酯的核糖核酸外切酶。我们报告了保守氨基酸取代对以下残基的影响：（i）His42、His48、Asp50、Arg52、His84和Tyr88，它们对核糖核酸酶和3'-磷酸酶活性都至关重要；（ii）Arg14、Asp15、Glu21和Glu82，它们对3'-磷酸酶活性至关重要，但对3'-核糖核苷去除不重要；（iii）Lys66和Arg76，它们选择性地参与3'-核糖核酸酶反应。结果表明了各个功能基团在金属结合和/或磷酸酯酶化学中的作用。
