The Rad50 signature motif: Essential to ATP binding and biological function
The Rad50 signature motif: Essential to ATP binding and biological function
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DOI：
10.1016/j.jmb.2003.11.026
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发表时间：
2004-01-23
影响因子：
5.6
通讯作者：
Paull, TT
中科院分区：
文献类型：
--
作者：
Moncalian, G;Lengsfeld, B;Paull, TT
DNA双链断裂的修复是所有生物体中必不可少的过程，并且需要进化上保守的蛋白质组装体的协调活动。其中最关键的一个是Mre 11/Rad 50（M/R）复合物，它存在于所有三个生物界中，但在生物化学水平上还没有得到很好的理解。以前的结构，从古细菌Rad 50同源物的分析阐明了催化核心的酶，ATP结合域相关的ABC转运蛋白家族的ATP酶。在这里，我们提出的晶体结构，Rad 50突变体S793 R。这种错义特征基序突变改变了Rad 50同源物和ABC ATP酶中保守的特征基序中的关键丝氨酸残基。S793 R突变类似于囊性纤维化跨膜传导调节因子（CFTR）中的突变S549 R，导致囊性纤维化。我们在这里表明，丝氨酸精氨酸的Rad 50蛋白的变化，防止ATP结合，并破坏其他ATP结合环之间的通信。这种结构变化反过来改变了Rad 50单体之间的连通，从而防止Rad 50二聚化。在人Rad 50基因中进行了等同的突变，并且所产生的突变蛋白确实与Mre 11和Nbs 1形成了复合物，但是在所有ATP依赖性酶活性中特异性地缺乏。这种标志性基序结构-功能同源性延伸到酵母，因为引入酿酒酵母RAD 50基因的相同突变产生了在体内DNA修复测定中未能补充rad 50缺失菌株的等位基因。这些结构和生化结果扩展了我们对Rad 50催化结构域的理解，并验证了使用签名基序突变体来测试Rad 50 ATP结合在不同生物体中的作用。(C)2003 Elsevier Ltd.保留所有权利。
The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/ Rad50 (M/R) complex, which is present in all three biological kingdoms, but, is not well-understood at the biochemical level. Previous structural, analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of, the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms. (C) 2003 Elsevier Ltd. All rights reserved.