Anton Brian P, Raleigh Elisabeth A
New England Biolabs, 32 Tozer Rd., Beverly, MA 01915, USA.
J Bacteriol. 2004 Sep;186(17):5699-707. doi: 10.1128/JB.186.17.5699-5707.2004.
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 linker 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 lambda 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 lambda 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.
McrA是大肠杆菌K-12中限制修饰的外源DNA的三种功能之一，对甲基化和羟甲基化底物均有影响。我们在此展示了通过使用GPS-LS插入扫描系统对McrA功能组织进行的首次系统分析。我们在McrA蛋白的46个独立位置收集了五个氨基酸的读框内插入，以及在20个独立位置的C端截短。每个突变体都针对甲基化和羟甲基化噬菌体（分别为M.HpaII修饰的λ噬菌体和T4gt噬菌体）的体内限制进行了检测，并针对在M.HpaII甲基化存在下大肠杆菌SOS应答的诱导进行了检测，这表明存在DNA损伤。我们的研究结果表明，存在一个N端DNA结合结构域和一个C端催化核酸酶结构域，它们由一个在很大程度上耐受氨基酸插入的连接区相连。功能性C端结构域造成的DNA损伤是限制噬菌体T4gt所必需的。N端结构域的破坏消除了对两种底物的限制。令人惊讶的是，保留N端结构域的截短突变不会介导DNA损伤（通过SOS诱导来衡量），但在体内仍能部分限制M.HpaII修饰的λ噬菌体。我们对这种“无损伤限制”以及在另一种修饰DNA限制功能的组分McrB中在体内观察到的类似现象提出了一个共同的解释。简而言之，我们提出该蛋白在λ基因组中的一个易损位置进行非生产性位点特异性结合，会在早期破坏噬菌体的发育程序。我们还鉴定出一个单一突变体，其在N端结构域有一个插入，它可以完全限制λ噬菌体，但根本不限制T4gt噬菌体。这个突变体可能在底物识别方面有选择性损伤，能区分甲基化和羟甲基化底物。该研究表明，当与有效的表型测试相结合时，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。
