Structure-Function Analysis of DNA Replication Initiation Factors Implicated in Disease

项目总结 DNA复制是一个高度保守的过程，对所有生命都是必不可少的。因为真核生物的基因组 它们规模庞大，从数千个复制源并行复制。对复制的适当调控 启动对于维持基因组的稳定性是必不可少的。癌基因激活失调复制启动 并使癌症中的细胞快速增殖。复制启动的关键调控因子在 癌症，而这些蛋白质的突变会导致罕见的遗传疾病。 然而，复制启动的分子机制仍然知之甚少，尤其是 在多细胞生物体中，出于两个主要原因：(1)复制启动涉及瞬时蛋白质-蛋白质和 利用现有的生化、遗传和结构来研究蛋白质-DNA的相互作用是非常具有挑战性的 方法；和(2)复制启动的最全面的研究已经在酵母中完成，然而关键是 在酵母和后生动物之间，复制起始调控因子的保守性很差。值得注意的是，一些人类 复制因子比酵母的复制因子大2-3倍，并且含有未知的新结构域 功能。 我的实验室最近开发了一个单分子成像平台来可视化复制启动 实时的。我们将使用这一强大的方法来(I)提供复制起始的定量描述； (Ii)了解这一过程如何从酵母转变为人类；(Iii)描述关键复制蛋白的作用 以及它们的后生动物特有的结构域；以及(Iv)理解为什么某些复制中的突变 启蒙因素导致疾病。我们的工作还将提供有价值的机制洞察癌细胞如何 通过过度激活复制启动来维持快速增殖，并告知复制启动因素 可用作新疗法的生物标记物或靶点。

PROJECT SUMMARY DNA replication is a highly conserved process essential for all life. Since eukaryotic genomes are very large, they are replicated in parallel from thousands of replication origins. Proper regulation of replication initiation is essential for maintaining genome stability. Oncogene activation dysregulates replication initiation and enables rapid cell proliferation in cancer. Key regulators of replication initiation are over-expressed in cancer, and mutations in these proteins cause rare genetic disorders. Yet, the molecular mechanisms that underlie replication initiation remain poorly understood, especially in multicellular organisms, for two main reasons: (1) replication initiation involves transient protein-protein and protein-DNA interactions that are very challenging to study using existing biochemical, genetic, and structural methods; and (2) the most comprehensive studies of replication initiation have been done in yeast, however key regulators of replication initiation are poorly conserved between yeast and metazoans. Notably, some human replication factors are 2-3x larger than their yeast counterparts and containing novel domains with unknown functions. My laboratory recently developed a single-molecule imaging platform to visualize replication initiation in real time. We will use this powerful approach to (i) provide a quantitative description of replication initiation; (ii) understand how this process changed from yeast to humans; (iii) delineate the role of key replication proteins and their domains that are unique to metazoans; and (iv) understand why mutations in some replication initiation factors cause disease. Our work will also provide valuable mechanistic insights into how cancer cells sustain rapid proliferation by over-activating replication initiation and inform how replication initiation factors may be used as biomarkers or targets for novel therapies.