Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Biochemistry. 2010 Jun 15;49(23):4872-83. doi: 10.1021/bi902021d.
DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.
DNA 解旋酶存在于所有生命领域，在 DNA 代谢过程中发挥着关键作用，如复制、修复、重组和转录。然而，迄今为止，DNA 解旋酶在分枝杆菌同源重组中的作用尚不清楚。在这项研究中，我们表明结核分枝杆菌 UvrD1 比非同源的耻垢分枝杆菌或大肠杆菌 RecA 蛋白更有效地抑制其同源 RecA 促进的链交换。缺乏解旋酶和 ATP 酶活性的结核分枝杆菌 UvrD1（Q276R）突变体能够阻断分枝杆菌 RecA 蛋白促进的链交换，但不能阻断大肠杆菌 RecA 的链交换。我们观察到结核分枝杆菌 UvrA 本身对大肠杆菌 RecA 促进的链交换没有明显影响，但会阻碍分枝杆菌 RecA 蛋白催化的反应。我们的数据还表明，结核分枝杆菌 UvrA 和 UvrD1 可以协同作用抑制分枝杆菌 RecA 蛋白促进的链交换。总之，这些发现提出了一种可能性，即 UvrD1 和 UvrA 可能在体内协同作用，以抵消 RecA 核蛋白丝的有害影响，和/或促进重组中间体的溶解。最后，我们提供了结核分枝杆菌 UvrD1 与 RecA 一方面以及 RecA 与 UvrA 另一方面之间物理相互作用的直接实验证据。这些观察结果与一种分子机制一致，即结核分枝杆菌 UvrA 和 UvrD1 协同作用，阻断同源和非同源 RecA 蛋白促进的 DNA 链交换。
