Structural Studies of MCM Complex

描述(申请人提供)：微小染色体维持蛋白(MCM)复合体的功能是一种解旋酶，解开DNA，为DNA复制提供模板，并在调节细胞生长方面发挥关键作用。MCM解旋酶被认为是细胞DNA复制的许可因素。真核生物和古菌的MCM蛋白形成六聚体环状复合体，分子量约为0.5-1.0兆道尔顿。尽管进行了广泛的努力，但对于MCM如何解离DNA，MCM如何在解离过程中与DNA相互作用，以及如何利用ATP来驱动解离所需的MCM的构象变化，我们仍存在知识差距。MCM在古生物和真核生物中是保守的。真核生物的MCM包含六个同系物，它们形成了异六聚体，可能还有更高阶的复合体。然而，一些古菌中的MCM复合体只含有一种MCM蛋白，可以组装成同源六聚体或十二聚体。因此，古生型MCM同源低聚物为理解MCM复合体的结构/功能提供了一个更简单的系统。该方案的目的是利用古生代MCM复合体作为一个模型系统来了解MCM复合体的结构/功能，特别是关于MCM蛋白的寡聚化、ATP引发的构象变化以及DNA结合和重塑功能的机制。这项研究将结合结构生物学、生物物理学和功能生物化学进行。所得数据将为理解复制叉处的DNA解卷机制提供有价值的信息。它还将为同源的真核MCM复合体提供结构/功能方面的见解，该复合体与细胞生长调控和癌症生物学具有高度相关性。与公共卫生相关：对所有活着的生物体来说，一个基本的过程是复制基因组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，解旋酶需要解压双螺旋DNA(DsDNA)，为复制过程中的子链合成提供单链DNA模板。MCM复合体是一种解离dsDNA的解旋酶，对DNA复制和细胞生长至关重要。我们建议结合结构生物学、生物物理学和功能生物化学的方法来研究这个重要的MCM解旋酶的结构和功能，以了解这种酶是如何利用ATP的能量来解开双螺旋DNA进行复制的。

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.