Davies G P, Powell L M, Webb J L, Cooper L P, Murray N E
Institute of Cell and Molecular Biology, Darwin Building, King's Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
Nucleic Acids Res. 1998 Nov 1;26(21):4828-36. doi: 10.1093/nar/26.21.4828.
For type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR). The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV. We provide evidence based on an analysis of Eco KI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes. We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs. Eco KI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage. The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity.
对于I型限制系统，最近确定的核苷酸序列预测了在限制而非修饰所必需的亚基（HsdR）中的保守氨基酸。这些保守序列强调了包含推定解旋酶的DEAD-box蛋白家族的特征基序，并且它们确定了基序IV的一个新候选者。我们基于对Eco KI的分析提供了证据，该证据支持DEAD-box基序与限制机制的相关性以及基序IV的新定义。新鉴定的基序IV内以及其他六个先前鉴定的DEAD-box基序中的氨基酸取代，而不是原始基序IV中的取代，赋予限制缺陷型表型。我们通过确定七个基序中每个基序的氨基酸取代产生的缺陷酶在体外允许的步骤，研究了DEAD-box基序与限制途径的相关性。从七个限制缺陷型突变体中纯化的Eco KI与未甲基化的靶序列结合，并且在AdoMet存在下，通过经历导致DNA切割的事件途径所必需的构象变化来响应ATP。这七种酶几乎没有或没有ATP酶活性和内切核酸酶活性，但它们保留了切割未修饰DNA的能力，尽管速率降低。因此，DNA链的切口可能是限制途径中必不可少的早期步骤，促进了DNA的ATP依赖性易位，特别是如果这涉及DNA解旋酶活性。
