Terry B J, Jack W E, Modrich P
J Biol Chem. 1985 Oct 25;260(24):13130-7.
The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).
已在几个含有两个EcoRI位点的DNA底物上研究了持续性EcoRI核酸内切酶的水解潜力，这些位点嵌入相同的序列环境中。对于一个388碱基对的环状DNA，其中两个识别位点相隔51个碱基对（较短距离）或337个碱基对（较长距离），在离子强度为0.059和0.13时，所有事件中分别有77%和34%涉及持续性水解。然而，对于线性底物（其中两个位点相隔51个碱基对），在这些离子强度下持续性作用的频率仅为42%和17%，该值是环状DNA观察值的一半。在离子强度为0.23时，在环状或线性底物上均未检测到持续性作用。这些发现表明，核酸内切酶对DNA的搜索是通过促进扩散进行的，在这种机制中，蛋白质通过与非特异性DNA位点相互作用来定位并离开其识别序列。我们认为，由于酶在切割线性分子产生的两种产物之间进行分配，线性底物上的持续性被限制为小环状底物的一半。考虑到这种拓扑效应，测得的持续性值意味着核酸内切酶可以在DNA区域内扩散，以定位并识别距初始结合位点50至300个碱基对远的EcoRI位点，在离子强度为0.059和0.13时最小搜索效率分别为80%和30%。持续性作用的高效率表明，在这种条件下，位置相关的搜索模式在该系统的促进扩散中起主要作用。当两个紧密间隔的EcoRI位点不对称地位于线性DNA末端附近时，识别出显著的位置效应也与该观点一致。核酸内切酶对位于更中心位置的识别序列表现出明显的偏好。这种偏好并不反映两个位点本身对切割的不同敏感性，而可以简单地解释为酶通过更中心位置的识别序列可利用的更大非特异性靶点优先进入。这些结论与之前对核酸内切酶在短距离内持续性的定性分析结果不同（Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501 - 513）。
