The type I restriction endonuclease EcoR1241, couples ATP hydrolysis to bidirectional DNA translocation
The type I restriction endonuclease EcoR1241, couples ATP hydrolysis to bidirectional DNA translocation
复制标题
DOI：
10.1016/j.jmb.2005.07.055
复制
发表时间：
2005-09-30
影响因子：
5.6
通讯作者：
Hurley, EM
中科院分区：
文献类型：
--
作者：
Bianco, PR;Hurley, EM
I型限制性内切核酸酶全酶含有甲基化酶（M）、限制性酶（R）和特异性酶（S）亚基，以M-2：R-2：S-1化学计量存在。这些酶与特定的DNA序列结合，并以ATP依赖性方式将dsDNA转运到锚定在识别序列上的全酶。一旦易位受阻，DNA限制，其功能是保护宿主细胞免受入侵的DNA，发生。易位和DNA切割由两个完全相反的R亚基提供。为了深入了解易位的机制，对EcoR 124 I的ATP酶活性进行了详细的表征。结果表明，识别序列结合后，ATP水解偶联，双向DNA易位的EcoR 124 I englomeration，与R-亚基瞬时脱离，平均每515 bp。由于R-亚基通过与甲基转移酶结合而保持与DNA紧密接近，因此维持2031（184）bp的宏观持续合成能力。ATP水解与易位的瞬时解偶联导致每个R亚基易位的每个碱基对水解3.1（+/-0.4）个ATP分子。这是ATP水解与dsDNA易位偶联的第一个明确的证明，尽管效率低下。一旦易位在超螺旋DNA上受阻，DNA就被切割。DNA切割使EcoR 124 I全酶部分可逆地失活，这解释了I型限制性内切酶的化学计量行为。失活的全酶在识别序列处保持与DNA结合，并立即释放新生末端。使用一种新型的基于荧光的实时测定法证明了新生末端的释放，该测定法利用了大肠杆菌RecBCD酶解开限制性dsDNA的能力。EcoR 124 I限制性DNA的RecBCD解旋揭示了限制性修饰和重组系统之间的协调，其功能是有效地破坏入侵DNA。此外，我们证明了EcoR 124 I的位移后，DNA切割的易位RecBCD酶，导致在恢复的催化功能EcoR 124 I。(c)2005爱思唯尔有限公司保留所有权利。
Type I restriction endonuclease holoenzymes contain methylase (M), restriction (R) and specificity (S) subunits, present in an M-2:R-2:S-1 stoichiometry. These enzymes bind to specific DNA sequences and translocate dsDNA in an ATP-dependent manner toward the holoenzyme anchored at the recognition sequence. Once translocation is impeded, DNA restriction, which functions to protect the host cell from invading DNA, takes place. Translocation and DNA cleavage are afforded by the two diametrically opposed R-subunits. To gain insight into the mechanism of translocation, a detailed characterization of the ATPase activity of EcoR124I was done. Results show that following recognition sequence binding, ATP hydrolysis-coupled, bidirectional DNA translocation by EcoR124I ensues, with the R-subunits transiently disengaging, on average, every 515 bp. Macroscopic processivity of 2031( 184) bp is maintained, as the R-subunits remain in close proximity to the DNA through association with the methyltransferase. Transient uncoupling of ATP hydrolysis from translocation results in 3.1(+/- 0.4) ATP molecules being hydrolyzed per base-pair translocated per R-subunit. This is the first clear demonstration of the coupling of ATP hydrolysis to dsDNA translocation, albeit inefficient. Once translocation is impeded on supercoiled DNA, the DNA is cleaved. DNA cleavage inactivates the EcoR124I holoenzyme partially and reversibly, which explains the stoichiometric behaviour of type I restriction enzymes. Inactivated holoenzyme remains bound to the DNA at the recognition sequence and immediately releases the nascent ends. The release of nascent ends was demonstrated using a novel, fluorescence-based, real-time assay that takes advantage of the ability of the Escherichia coli RecBCD enzyme to unwind restricted dsDNA. The resulting unwinding of EcoR124I-restricted DNA by RecBCD reveals coordination between the restriction-modification and recombination systems that functions to destroy invading DNA efficiently. In addition, we demonstrate the displacement of EcoR124I following DNA cleavage by the translocating RecBCD enzyme, resulting in the restoration of catalytic function to EcoR124I. (c) 2005 Elsevier Ltd. All rights reserved.