Department of Chemistry, University of Toledo, Toledo OH, USA.
Virol J. 2010 Dec 3;7:359. doi: 10.1186/1743-422X-7-359.
The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.
噬菌体 T4 编码了 10 种蛋白质，统称为复制体，负责噬菌体基因组的复制。复制体蛋白可以细分为三种活性；负责复制 DNA 的复制酶、负责解旋和冈崎片段起始的原核蛋白、负责冈崎修复的蛋白。复制酶包括 gp43 DNA 聚合酶、gp45 进程性夹、gp44/62 夹加载复合物和 gp32 单链 DNA 结合蛋白。原核蛋白包括 gp41 六聚体解旋酶、gp61 引物酶和 gp59 解旋酶加载蛋白。RNaseH，一种 5'到 3'外切核酸酶和 T4 DNA 连接酶，构成了冈崎修复所必需的活性。T4 提供了一个 DNA 复制的模型系统。因此，人们付出了巨大的努力来解决这些复制体蛋白的晶体结构。在这篇综述中，我们讨论了现有的结构，并在 T4 结构不可用时提供了与相关蛋白的比较。十个全长 T4 复制体蛋白中有三个已经确定；gp59 解旋酶加载蛋白、RNase H 和 gp45 进程性夹。T4 核心蛋白 gp32 和来自 T4 相关噬菌体 RB69 的两种蛋白，gp43 聚合酶和 gp45 夹也已解决。T4 gp44/62 加载器尚未结晶，但与大肠杆菌γ复合物进行了比较。T4 gp41 解旋酶、gp61 引物酶和 T4 DNA 连接酶的结构尚不清楚，而是讨论了噬菌体 T7 蛋白的结构。为了更好地理解 T4 DNA 复制的功能，需要对蛋白质和 DNA 底物之间的复合物进行深入的结构分析。gp43 聚合酶结合 DNA 引物模板、RNase H 结合分叉 DNA 底物、gp43 聚合酶结合 gp32 蛋白和 RNase H 结合 gp32 蛋白的复合物已通过晶体学确定。复合物的制备和结晶是一个重大挑战。我们讨论了替代方法，如小角度 X 射线和中子散射，以产生建模大分子组装的分子包络。
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