Structure and function of the ACMV geminivirus replication initiator protein

植物双生病毒的复制相关蛋白（Rep）是一种多功能蛋白，其通过滚环复制启动病毒复制并与多种宿主细胞因子相互作用。此外，双生病毒Rep蛋白具有ATP酶/解旋酶功能。非洲木薯花叶病毒Rep蛋白将在裂殖酵母（裂殖酵母）中异位表达，并从细胞提取物中纯化用于进一步分析。通过使用电子显微镜（EM）和尺寸排阻色谱（SEC），将评估在不同阳离子以及腺苷核苷酸和DNA存在下Rep蛋白寡聚体的形成。这些实验旨在分析具有不同数量单体的Rep复合物是否可能涉及Rep蛋白的不同功能（例如，用于启动复制的核酸内切裂解，解旋酶）。基于这些分析，将通过电子显微镜和图像处理确定功能性Rep寡聚体的三维结构。这些结构将有助于在分子水平上理解Rep蛋白中结合的不同功能。

The replication-associated protein (Rep) of plant geminiviruses is a multifunctional protein necessary for initiation of viral replication by rolling circle replication and for interaction with various host cell factors. In addition, geminivirus Rep proteins possess ATPase/helicase function. African cassava mosaic virus Rep protein will be ectopically expressed in fission yeast (Schizosaccharomyces pombe) and purified from cell extracts for further analysis. By using electron microscopy (EM) and size exclusion chromatography (SEC) the formation of Rep protein oligomers in the presence of different cations as well as adenosine nucleotides and DNA will be assessed. These experiments are aimed to dissect if Rep complexes with different numbers of monomers might be involved for different functions of the Rep protein (e.g. endonucleolytic cleavage for initiation of replication, helicase). Based on these analyses, three dimensional structures of the functional Rep oligomers will be determined by electron microscopy and image processing. The structures will help to understand the different functions that are combined in the Rep protein on a molecular level.