Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease
Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease
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DOI：
10.1128/jb.186.17.5699-5707.2004
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发表时间：
2004-09-01
影响因子：
3.2
通讯作者：
Raleigh, EA
中科院分区：
文献类型：
--
作者：
Anton, BP;Raleigh, EA
McrA是限制大肠杆菌K-12中修饰的外源DNA的三种功能之一，影响甲基化和羟甲基化底物。我们在这里提出的第一个系统的分析McrA的功能组织通过使用GPS-LS插入扫描系统。我们收集了5个氨基酸在46个独立的位置和C-末端截短在20个独立的位置在McrA蛋白的框内插入。测定每种突变体对甲基化和羟甲基化噬菌体（分别为M. HpaII修饰的λ和T4 gt）的体内限制性以及对大肠杆菌的诱导。在存在M.HpaII甲基化的情况下，大肠杆菌SOS响应，指示DNA损伤。我们的研究结果表明存在一个N-末端DNA结合结构域和一个C-末端催化核酸酶结构域连接的lint：er区域很大程度上容忍氨基酸插入。限制噬菌体T4 gt需要由功能性C-末端结构域造成的DNA损伤。N-末端结构域的破坏消除了对两种底物的限制。令人惊讶的是，截断突变，备用的N-末端结构域不介导DNA损伤，SOS诱导测量，但仍然部分限制M. HpaII修饰的X在体内。我们提出了一个共同的解释，这种“限制无损伤”和类似的观察在体内与McrB，另一个修改的DNA限制功能的组成部分。简而言之，我们提出，非生产性的位点特异性结合的蛋白质在λ基因组中的一个脆弱的位置破坏了噬菌体的发展计划在早期阶段。我们还确定了一个单一的突变体，携带插入的N-末端结构域，这可以完全限制X，但不限制T4 gt在所有。该突变体可能在底物识别、区分甲基化和羟甲基化底物方面具有选择性损伤。该研究表明，技术上简单的插入扫描方法可以提供丰富的功能信息来源时，结合有效的表型测试。
McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified lambda and T4gt, respectively) and for induction of the E. coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a lint:er region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified X in vivo. We suggest a common explanation for this "restriction without damage" and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the lambda genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict X but did not restrict T4gt at all. This mutant may have a selective impairment in substrate, recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.