Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong.
BMC Mol Biol. 2011 Apr 18;12:16. doi: 10.1186/1471-2199-12-16.
SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.
SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.
The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.
SXT 是一种整合共轭元件（ICE），最初从霍乱弧菌中分离出来，霍乱弧菌是引起霍乱的细菌病原体。它在其约 100kb 大小的圆形双链 DNA（dsDNA）基因组上拥有多种抗生素和重金属抗性基因，并作为抗性基因在易感细菌群体中水平转移的有效载体。在这里，我们描述了 SXT 遗传元件上编码的碱性外切酶（S066，SXT-Exo）和单链退火蛋白（S065，SXT-Bet）的活性，它们分别与噬菌体 lambda 的 Exo 和 Bet 具有显著的序列同源性。
SXT-Exo 具有降解线性 dsDNA 和单链 DNA（ssDNA）分子的能力，但没有检测到内切酶或切口活性。SXT-Exo 在溶液中采用稳定的三聚体排列，其外切酶活性在 pH8.2 时最佳，并且基本上需要 Mn2+或 Mg2+离子。与 lambda-Exo 相似，SXT-Exo 以高度连续的方式从 5'到 3'极性水解 dsDNA，并以显著更高的效率消化具有 5'磷酸化末端的 DNA 底物，而缺乏 5'磷酸基团的 DNA 底物则不然。值得注意的是，添加 lambda-Bet、SXT-Bet 或 SXT 遗传元件（S064，SXT-Ssb）上编码的单链 DNA 结合蛋白可刺激 SXT-Exo 和 lambda-Exo 的 dsDNA 外切酶活性。当在大肠杆菌细胞中共表达时，SXT-Bet 和 SXT-Exo 介导由 PCR 生成的 dsDNA 片段与染色体之间的同源重组，类似于 RecET 和 lambda-Bet/Exo。
SXT-Exo 蛋白的活性表明它能够切除线性化 dsDNA 分子的末端，形成部分 ssDNA 底物，供与之配对的 SXT-Bet 单链退火蛋白使用。因此，SXT-Exo 和 SXT-Bet 可能一起作用，通过促进同源 DNA 重组事件，修复或处理感染霍乱弧菌细胞内的 SXT 遗传元件。本研究结果显著扩展了我们对病毒/噬菌体来源的碱性外切酶和单链退火蛋白的性质和活性的一般认识，并将有助于合理开发细菌重组系统。
