Au K G, Welsh K, Modrich P
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1992 Jun 15;267(17):12142-8.
Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K. M., Lu, A.-L., Clark, S., and Modrich, P. (1987) J. Biol. Chem. 262, 15624-15629). MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA. The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T greater than G-G greater than A-C greater than C-C), and activated MutH responds to the state of DNA adenine methylation. Incision of an unmethylated strand occurs immediately 5' to a d(GATC) sequence, leaving 5' phosphate and 3' hydroxy termini (pN decreases pGpAp-TpC). Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine. The mechanism of activation apparently requires ATP hydrolysis since adenosine-5'-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP. The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3' or 5' to the mismatch on an unmethylated strand. However, activation is sensitive to DNA topology. Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule. MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate. We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.
大肠杆菌MutH具有一种极其微弱的d(GATC)核酸内切酶，它对该序列的甲基化状态有反应（威尔士，K.M.，卢，A.-L.，克拉克，S.，和莫德里奇，P.（1987年）《生物化学杂志》262，15624 - 15629）。MutH核酸内切酶在一个需要MutS、MutL、ATP和Mg2+的反应中被激活，并且依赖于DNA内错配的存在。激活程度与特定错配接受甲基导向修复的效率相关（G - T大于G - G大于A - C大于C - C），并且被激活的MutH对DNA腺嘌呤甲基化状态有反应。未甲基化链的切割立即发生在d(GATC)序列的5'端，留下5'磷酸和3'羟基末端（pN降低pGpAp - TpC）。未甲基化的d(GATC)位点会被激活的MutH进行双链切割，这种效应可能解释了2 - 氨基嘌呤对dam - 突变体的致死作用。激活机制显然需要ATP水解，因为腺苷 - 5'-O-(3 - 硫代三磷酸)不仅不能支持该反应，而且还会抑制ATP促进的激活。该过程没有绝对的极性，因为被激活的核酸酶对未甲基化链上错配位点3'或5'端的d(GATC)位点都能进行切割。然而，激活对DNA拓扑结构敏感。环状异源双链体比线性分子是更好的底物，并且后一类DNA的活性取决于错配和d(GATC)位点在分子内的位置。MutH激活由一个6千碱基的线性异源双链体支持，其中错配和d(GATC)位点位于中心位置且相隔1千碱基，但一个相关分子，其中这两个位点位于DNA相对两端附近，作为底物基本上无活性。我们得出结论，MutH激活代表甲基导向修复的起始阶段，并表明错配与d(GATC)位点的相互作用是由MutS与错配碱基对结合引发的，随后一个或多个Mut蛋白沿螺旋进行ATP依赖的易位，导致在错配两侧的d(GATC)序列处切割。
