Structural Studies of MCM Complex

描述（由申请方提供）：微小染色体维持蛋白（MCM）复合物作为解旋酶发挥功能，解旋DNA以提供DNA复制模板，并在调节细胞生长中发挥关键作用。MCM解旋酶被认为是细胞DNA复制的许可因子。来自真核生物和古细菌的MCM蛋白形成六聚体环复合物，MWt约为0.5-1.0 Mega Dolton。尽管做了大量的努力，但我们对MCM如何解旋DNA、MCM在解旋过程中如何与DNA相互作用以及如何利用ATP来驱动MCM解旋所需的构象变化的理解存在知识缺口。MCM在古生菌和真核生物中是保守的。真核MCM含有六个形成异源六聚体和可能更高阶复合物的同系物。然而，在一些古细菌中的MCM复合物仅包含一个MCM蛋白，其可以组装成同源六聚体或十二聚体。因此，古细菌MCM同源寡聚物为理解MCM复合物的结构/功能提供了一个更简单的系统。本研究的目的是利用古菌MCM复合物作为模型系统来了解MCM复合物的结构/功能，特别强调MCM蛋白的寡聚化、ATP引发的构象变化以及DNA结合和重构功能的机制。将结构生物学、生物物理学和功能生物化学相结合进行研究。所得数据将为理解复制叉处的DNA解旋机制提供有价值的信息。它还将提供同源真核MCM复合物的结构/功能的见解，这与细胞生长调节和癌症生物学具有高度相关性。公共卫生相关性：所有生物体的一个基本过程是复制基因组DNA（或DNA复制），以确保正确的遗传信息传递。为了复制基因组DNA，需要解旋酶来解压缩双螺旋DNA（dsDNA）以在复制过程中为子链合成提供ssDNA模板。MCM复合物是解旋dsDNA的解旋酶，对DNA复制和细胞生长至关重要。我们建议使用包括结构生物学、生物物理学和功能生物化学在内的方法来研究这种重要的MCM解旋酶的结构/功能，以了解这种酶如何利用ATP的能量来解开双螺旋DNA进行复制。

DESCRIPTION (provided by applicant): Minichromosome maintenance protein (MCM) complex functions as a helicase that unwinds DNA to provide template for DNA replication and plays a critical role in regulating cell growth. MCM helicases are considered to be a licensing factor for cellular DNA replication. MCM proteins from eukaryotes and archaea form hexamers ring complex, with MWt approximating 0.5-1.0 Mega Dolton. Despite extensive efforts, gap of knowledge exists in our understanding of how MCM unwinds DNA, how MCM interacts with DNA during unwinding, and how ATP is utilized to drive the conformational changes of MCM needed for unwinding. MCM are conserved in archaea and eukaryotes. Eukaryotic MCM contains six homologs that form hetero-hexamer and possibly higher order complexes. However, the MCM complex in some archaea contains only one MCM protein that can assemble into homo-hexamers or dodecamers. Thus, archaeal MCM homo-oligomers provide a simpler system for understanding the structure/function of MCM complex. The goal of this proposal is to use archaeal MCM complexes as a model system to understand the structure/functions of MCM complex, with particular emphasis on the mechanisms regarding oligomerization, ATP-triggered conformational changes, and DNA binding and remodeling functions of MCM proteins. A combination of structural biology, biophysics, and functional biochemistry will be employed for the study. The resulting data will provide valuable information for understanding the DNA unwinding mechanism at the replication fork. It will also provide structural/functional insights for the homologous eukaryotic MCM complex, which bears high relevance to cell growth regulation and cancer biology. PUBLIC HEALTH RELEVANCE: One essential process for all living organisms is duplicate the genomic DNA (or DNA replication) to ensure correct genetic information passage. To duplicate genomic DNA, helicases is required to unzip the double helix DNA (dsDNA) to provide ssDNA template for daughter strand synthesis in the replication process. MCM complex is such a helicase that unwinds dsDNA and is critical for DNA replication and cell growth. We propose to study the structure/function of this important MCM helicase using a combination of approaches including structural biology, biophysics, and functional biochemistry, in order to understand how this enzyme utilizes the energy of ATP to unwind the double helix DNA for replication.