Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy
Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy
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DOI：
10.1074/jbc.c200129200
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发表时间：
2002-05-03
影响因子：
4.8
通讯作者：
Sancar, A
中科院分区：
文献类型：
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作者：
Griffith, JD;Lindsey-Boltz, LA;Sancar, A
人检查点Rad蛋白被认为在DNA损伤检查点反应途径中起损伤传感器的作用。检查点蛋白hRad 9、hHus 1和hRad 1与复制持续性因子增殖细胞核抗原（PCNA）具有有限的同源性，而hRad 17与复制因子C（RFC）具有同源性。这样的观察结果导致了这样的建议，这些检查点Rad蛋白可能在检查点控制期间与它们的复制对应物功能相似。我们纯化了两个复合物形成的检查点拉德蛋白，并研究了它们的结构，使用电子显微镜制备方法，其中的复合物从甘油溶液喷雾到非常薄的碳箔上，在真空中用钨装饰，并在低电压下成像。我们发现hRad 9，hHus 1和hRad 1蛋白质形成了一个三聚体环结构（检查点9-1-1复合物），让人联想到PCNA环。类似地，我们发现hRad 17与四个RFC小亚基（hRad 17-RFC）形成具有深凹槽或裂缝的异五聚体复合物，并且类似于RFC夹装载器。因此，我们的研究结果表明检查点拉德复合物和PCNA和RFC复制因子之间的结构相似性，从而为这些复合物提出类似功能的模型提供进一步的支持。
Human checkpoint Rad proteins are thought to function as damage sensors in the DNA damage checkpoint response pathway. The checkpoint proteins hRad9, hHus1, and hRad1 have limited homology to the replication processivity factor proliferating cell nuclear antigen (PCNA), and hRad17 has homology to replication factor C (RFC). Such observations have led to the proposal that these checkpoint Rad proteins may function similarly to their replication counterparts during checkpoint control. We purified two complexes formed by the checkpoint Rad proteins and investigated their structures using an electron microscopic preparative method in which the complexes are sprayed from a glycerol solution onto very thin carbon foils, decorated in vacuo with tungsten, and imaged at low voltage. We found that the hRad9, hHus1, and hRad1 proteins make a trimeric ring structure (checkpoint 9-1-1 complex) reminiscent of the PCNA ring. Similarly we found that hRad17 makes a heteropentameric complex with the four RFC small subunits (hRad17-RFC) with a deep groove or cleft and is similar to the RFC clamp loader. Therefore, our results demonstrate structural similarity between the checkpoint Rad complexes and the PCNA and RFC replication factors and thus provide further support for models proposing analogous functions for these complexes.