首页> 外文期刊>Journal of Molecular Biology >Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.
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Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

机译:变构开关残基组氨酸195在维持噬菌体T4 UvsX重组酶突触前丝中活性位点不对称中的作用。

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摘要

Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPaseactive sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.
机译:高度保守的RecA / Rad51家族的重组酶在同源重组和DNA双链断裂修复中起着核心作用。 RecA / Rad51酶在单链DNA(ssDNA)上形成突触前丝,并被变构激活以催化ATPase和DNA链交换反应。信息是通过高度保守的谷氨酰胺残基(大肠杆菌RecA中的Gln194)在DNA和ATP结合位点之间传递的,该残基充当了变构开关。 T4 UvsX蛋白是一个不同的RecA直向同源物,在变构开关位置含有组氨酸(His195)代替谷氨酰胺。 UvsX和RecA催化相似的链交换反应,但其他性质不同。 UvsX产生ADP和AMP作为其ssDNA依赖的ATPase活性的产物-这种特性在表征的重组酶中是独特的。 ssDNA依赖的ATP水解反应动力学的详细信息表明,UvsX-ssDNA突触前丝不对称,并且包含两类ATP酶活性位点:一类产生ADP,另一类产生AMP。与野生型UvsX相比,UvsX-H195Q和UvsX-H195A突变体均表现出更强的ssDNA依赖性ATPase活性,较低的协同性和显着较高的ADP / AMP产物比率,因此可通过His195位置的突变来减少活性位点的不对称性。活性位点不对称性的降低与H195Q和H195A突变体中ssDNA结合亲和力和DNA链交换活性的降低密切相关。这些和其他结果支持一个模型,其中变构开关残基His195控制在DNA链交换中活跃的UvsX-ssDNA细丝的不对称构象的形成。讨论了我们的发现对UvsX重组功能和RecA功能的影响。

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