78 Structural studies of rolling circle replication initiator proteins
78 Structural studies of rolling circle replication initiator proteins
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78 滚环复制起始蛋白的结构研究
DOI：
10.1080/07391102.2013.786512
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发表时间：
2013
影响因子：
4.4
通讯作者：
Carr S
中科院分区：
文献类型：
--
作者：
Carr S
pT181家族的质粒通过滚动循环机制进行复制。该过程由质粒编码的Rep启动蛋白启动，该蛋白具有序列特异性的DNA切割和宗教活性。质粒复制起点被Rep标记，它通过一个活性位点酪氨酸与一条DNA链共价结合，启动滚动环复制，并在循环结束时重新生成该链。Rep蛋白也与PcrA解旋酶结合，形成高度加工的复合体。我们从嗜脂嗜热地杆菌（geobacillus stearotherophilus, Gst）的隐质粒pSTK1和金黄色葡萄球菌（staphylococcus aureus, Sau）的RepD变体中确定了Rep蛋白的结构，首次获得了这类启动物的结构信息。从pSTK1中克隆和表达指定的269 aa Rep产物未能产生可溶的活性蛋白。然而，从上游任意点表达产生一个细长的产物，能够放松质粒底物，编码来自pSTK1的反向重复序列，类似于pT181家族的复制起源。该产物和一个31 kDa的蛋白水解衍生的片段都显示了cognateGstPcrA解旋酶的激活，但没有显示s的激活。葡萄球菌。gstrep的31 kDa片段的晶体结构以2.3 Å的分辨率进行了解析，显示出一个不寻常的环状二聚体，其孔径为20 Å。环的内表面主要由18链β片组成，而外表面则装饰有18链α-螺旋。这种蛋白质代表了一种新的折叠；然而，延长的薄片确实表现出与tata结合蛋白和转录因子IID中观察到的相似之处。活性位点Tyr179残基，每个亚基一个，在孔中间隔26 Å，附近有一个催化镁离子，由三个羧酸侧链配位。以gstrep为模型，通过分子置换的方法对RepDE、RepDN和RepDC三种theSauRep变异体的晶体结构进行了解析，发现它们具有相似的结构特征。对滚动循环复制机制的影响将讨论在广泛的功能数据可用于saurepd。
Plasmids of the pT181 family replicate by a rolling-circle mechanism. The process is initiated by a plasmid-encoded Rep initiator protein, which has sequence-specific DNA nicking and religation activity. The plasmid replication origin is nicked by Rep, which binds covalently to one DNA strand via an active site tyrosine, initiating rolling circle replication and religating the strand at the end of the cycle. Rep proteins also associate with PcrA helicase to form a highly processive complex. We have determined the structure of the Rep protein from cryptic plasmid pSTK1 ofGeobacillus stearothermophilus(Gst), and several variants of RepD fromStaphylococcus aureus(Sau),representing the first structural information on this class of initiators. Cloning and expression of the designated 269 aa Rep product from pSTK1 failed to yield soluble, active protein. However, expression from an arbitrary point upstream yielded an elongated product capable of relaxing plasmid substrates encoding an inverted repeat sequence from pSTK1 which resembles the replication origin of the pT181 family. Both this product and a 31 kDa proteolytically derived fragment omitting the C-terminus additionally display activation of the cognateGstPcrA helicase, but not that ofS. aureus.The crystal structure of the 31 kDa fragment ofGstRep has been solved at 2.3 Å resolution, showing an unusual ring-shaped dimer with a 20 Å diameter pore. The inner surface of the ring is largely formed by an 18-stranded β-sheet, while the outer surface is decorated with 18α-helices. The protein represents a novel fold; however, the extended sheet does exhibit some similarities to those observed in both TATA-binding protein and transcription factor IID. The active site Tyr179 residues, one from each subunit, lie 26 Å apart across the pore, with a nearby catalytic magnesium ion coordinated by three carboxylate side-chains. Crystal structures for theSauRep variants termed RepDE, RepDN and RepDC have been solved by molecular repacement using theGstRep as a model, and show similar structural features. The implications for the mechanism of rolling circle replication will be discussed in the light of extensive functional data available forSauRepD.