Rao D E C S, Luo Yu
Department of Biochemistry, University of Saskatchewan, 2D01 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5.
Protein Pept Lett. 2014 Jul;21(7):679-87. doi: 10.2174/0929866521666140320103512.
RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein's ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA's pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase- inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.
RadA是人类重组酶Rad51在古菌中的同源物。这种重组酶超家族还包括真核生物减数分裂特异性的DMC1以及亲缘关系较远的细菌RecA，它们在ATP存在的情况下能在单链DNA上形成细丝，并促进单链DNA与同源双链DNA之间的链交换反应。由于其易于获得晶体以及与真核生物同源物的相似性（序列同一性>40%），我们研究了来自沃氏甲烷球菌的RadA（MvRadA），将其作为理解同源链交换分子机制的结构模型。在此我们表明，该蛋白的ATP酶活性和链交换活性在pH 6.0时最低。有趣的是，MvRadA对pH的依赖性与人类Rad51的特性相似，但与经过充分研究的大肠杆菌RecA不同。随后在pH 6.0时确定的结构揭示了一些特征，表明其为一种具有无序L2环的ATP酶无活性形式。与之前在pH 7.5时确定的ATP酶活性形式进行比较表明，在酸性pH条件下，ATP酶活性构象的稳定性降低。我们将这些结果解读为进一步表明，促进ATP水解以及单链和双链DNA之间的链交换需要DNA结合L2区域具有类似于之前观察到的ATP酶活性构象中的有序排列。移动L2区域中的His-276被观察到部分负责MvRadA的pH依赖性活性。
