Eggleston A K, Kowalczykowski S C
Department of Cell, Molecular & Structural Biology, Northwestern University Medical School, Chicago, IL 60611.
J Mol Biol. 1993 Jun 5;231(3):605-20. doi: 10.1006/jmbi.1993.1313.
RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination. Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.
大肠杆菌的RecBCD酶是一种DNA解旋酶，它还具有ATP依赖性核酸酶活性。我们纯化了一种突变型recBCD酶，命名为recB2109CD酶，并在体外检测了该蛋白的核酸酶活性，以确定这些活性的任何改变是否导致了recB2109菌株的重组缺陷表型。recB2109CD酶具有与野生型recBCD酶相关的所有非特异性核酸酶活性（双链DNA外切核酸酶和单链DNA外切及内切核酸酶），尽管相对于野生型酶，它们降低了约2至3倍。与野生型酶相比，recB2109CD酶的ATP依赖性双链DNA外切核酸酶活性需要显著更高的ATP浓度才能达到最佳活性。两种酶的ATP非依赖性单链DNA内切核酸酶活性相似，但突变酶的ATP刺激的单链DNA内切核酸酶和ATP依赖性单链DNA外切核酸酶活性相对于野生型recBCD酶有所降低。尽管recB2109CD酶能够非特异性地降解线性双链DNA，但它在chi位点缺乏序列特异性切口活性，而chi位点是基因重组的热点。由于这种与chi的相互作用会显著减弱野生型recBCD酶的非特异性双链DNA外切核酸酶活性，这些结果表明突变酶的非特异性双链DNA外切核酸酶活性不能被减弱，结果是无法产生适合重组的DNA底物。
