Uson Maria Loressa, Ghosh Shreya, Shuman Stewart
Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA.
Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA
J Bacteriol. 2017 Aug 8;199(17). doi: 10.1128/JB.00304-17. Print 2017 Sep 1.
We characterize FenA as a manganese-dependent 5'-flap endonuclease homologous to the 5'-exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' single strand is eliminated to create a 5'-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3'-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5' App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD-dependent DNA ligases and especially by mycobacterial DNA ligases D and C. Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in This study identifies FenA as a stand-alone endonuclease homologous to the 5'-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5' flaps, 5' nicks, and 5' App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of Pol1 is also shown to be a flap endonuclease.
我们将FenA鉴定为一种依赖锰的5'-翼瓣内切核酸酶，与DNA聚合酶I的5'-外切核酸酶同源。FenA在双链片段的第一个和第二个核苷酸之间切割带切口的5'翼瓣，产生一个1个核苷酸缺口的DNA，然后以二核苷酸步骤进一步切除。FenA在Y型翼瓣或切口处的初始切割发生在双链的第一个和第二个核苷酸之间。然而，当模板3'单链被去除以形成5'-尾双链时，FenA切割位点转移到第二个和第三个核苷酸之间。具有可移动连接的双翼瓣底物（模拟缺口修复期间有限的链置换合成）优先被切割为1个核苷酸的3'-翼瓣异构体，与静态双翼瓣相比，可切割的磷酸二酯移位了一个核苷酸。FenA有效地去除了流产的切口连接反应中间体的5' App(dN)末端，从而突出了FenA作为修复此类损伤的因子，这些损伤在多种情况下由细菌NAD依赖性DNA连接酶，特别是分枝杆菌DNA连接酶D和C形成。结构特异性DNA内切核酸酶参与细菌DNA复制、修复和重组，但对此类核酸酶的种类和催化功能知之甚少。本研究鉴定FenA为一种与分枝杆菌DNA聚合酶1的5'-外切核酸酶结构域同源的独立内切核酸酶。FenA切割5'翼瓣、5'切口和流产的切口连接的5' App(dN)中间体。还显示Pol1分离的N末端结构域是一种翼瓣内切核酸酶。
