Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.
Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.
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从纯化的亚基中重建大肠杆菌 RecBCD 全酶的活性。
DOI：
10.1016/s0021-9258(18)42249-1
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
P. T. Emmerson
中科院分区：
文献类型：
--
作者：
C. Masterson;P. Boehmer;F. McDonald;S. Chaudhuri;I. Hickson;P. T. Emmerson
大肠杆菌RecBCD全酶和单个组成亚基已从高产菌株中纯化。纯化的RecBCD全酶具有约330 kDa的天然分子量，表明异源三聚体亚基组装。RecB、RecC和RecD亚基可以在体外结合，产生核酸酶、解旋酶、ATP酶和Chi特异性核酸内切酶活性，这些活性与RecBCD全酶的活性无法区分。在重构的RecB + C + D酶非常有活性的浓度下，各个RecB、RecC或RecD亚基都不具有可容易检测的全酶活性，除了RecB蛋白，其先前已显示出DNA依赖性ATP酶活性（Hickson，I. D、罗布森角，澳-地N.，Atkinson，K. E、赫顿湖，和Emmerson，P. T.（1985）J.Biol.Chem.260，1224-1229）。在较高浓度和较短的DNA底物下，重构的RecBC蛋白表现出低水平的解旋酶和核酸外切酶活性。
The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.