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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >Characterization of Mycobacterium leprae RecA Intein a LAGLIDADG Homing Endonuclease Reveals a Unique Mode of DNA Binding Helical Distortion and Cleavage Compared with a Canonical LAGLIDADG Homing Endonuclease
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Characterization of Mycobacterium leprae RecA Intein a LAGLIDADG Homing Endonuclease Reveals a Unique Mode of DNA Binding Helical Distortion and Cleavage Compared with a Canonical LAGLIDADG Homing Endonuclease

机译：LAGLIDADG归巢核酸内切酶麻风杆菌RecA Intein的表征揭示了与典型LAGLIDADG归巢核酸内切酶相比独特的DNA结合螺旋变形和裂解模式

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摘要

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of the M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches, including four complementary footprinting assays such as DNase I, copper-phenanthroline, methylation protection, and KMnO4, enhancement of 2-aminopurine fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site, and generates two staggered double strand breaks. Taken together, these results implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of the LAGLIDADG family of homing endonucleases.
机译：麻风分枝杆菌经历了还原性进化，只留下了极少的必需基因集，但在其四个基因中保留了中间序列，这暗示着它们对麻风杆菌的生存至关重要。已经发现单个框内插入序列嵌入其recA基因内。麻风分枝杆菌recA插入序列与已知插入序列的比较表明，它具有作为LAGLIDADG型归巢核酸内切酶所必需的共有氨基酸序列。鉴于麻风杆菌中大量的基因衰退和功能丧失，我们试图研究其recA干预序列是否编码催化活性的归巢核酸内切酶。在这里，我们显示纯化的麻风杆菌RecA内含肽（PI-MleI）与同源DNA结合，并在其他二价阳离子，Mg 2 + 或Mn 2+ < / sup>。多种方法的组合，包括DNase I，铜-菲咯啉，甲基化保护和KMnO4等四种互补的足迹测定法，2-氨基嘌呤荧光的增强以及切割位点的作图显示，PI-MleI与与其插入侧翼的同源DNA结合位点，在切割位点引起螺旋形扭曲，并产生两个交错的双链断裂。综上所述，这些结果暗示PI-MleI具有模块化结构，该结构具有用于DNA靶标识别和切割的独立结构域，每个结构域具有不同的序列偏好。从生物学的角度出发，很容易推测我们的发现对理解归巢核酸内切酶LAGLIDADG家族的进化具有启示意义。

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