Murante R S, Huang L, Turchi J J, Bambara R A
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
J Biol Chem. 1994 Jan 14;269(2):1191-6.
The catalytic activity of the calf thymus 5'- to 3'-exonuclease was measured on substrates consisting of two primers annealed adjacent to each other on a template. Exonucleolytic degradation of the downstream primer is very slow if the primers are separated by a gap of one nucleotide or if no upstream primer is present. When only a nick separates the primers, degradation is rapid. This suggests that the nuclease is designed to work with calf DNA polymerases such that synthesis from an upstream primer creates the favored nuclease substrate. Nuclease action then destroys the substrate, but it is regenerated by further polymerization. This process, termed nick translation, is necessary for both DNA replication and repair. If the downstream primer has an unannealed 5'-region, that region is removed by an endonuclease activity residing in the same enzyme. Efficient endonuclease action also requires an upstream primer that is annealed such that its 3'-end is directly adjacent to the annealed region of the downstream primer. This reaction is likely to be important for removal of DNA segments that are damaged such that exonuclease cleavage of the damaged site is not possible.
在由两条在模板上彼此相邻退火的引物组成的底物上，测定了小牛胸腺5'至3'核酸外切酶的催化活性。如果引物之间间隔一个核苷酸的缺口，或者不存在上游引物，下游引物的核酸外切酶降解非常缓慢。当引物之间仅存在一个切口时，降解迅速。这表明该核酸酶设计用于与小牛DNA聚合酶协同工作，使得从上游引物进行的合成产生有利的核酸酶底物。然后核酸酶作用破坏底物，但通过进一步聚合使其再生。这个过程称为切口平移，对于DNA复制和修复都是必需的。如果下游引物有一个未退火的5'区域，该区域会被存在于同一酶中的内切核酸酶活性去除。高效的内切核酸酶作用也需要一个退火的上游引物，使其3'末端直接与下游引物的退火区域相邻。该反应可能对于去除受损的DNA片段很重要，因为受损位点的核酸外切酶切割是不可能的。
