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

项目摘要 DNA复制是一个高度保守的过程，对所有生命都至关重要。因为真核生物的基因组 大的时候，它们从数千个复制起点并行复制。正确调节复制 起始对于维持基因组稳定性是必需的。癌基因激活失调复制起始 并使癌症中的细胞快速增殖。复制起始的关键调节因子在细胞中过表达， 癌症，这些蛋白质的突变会导致罕见的遗传疾病。 然而，复制起始的分子机制仍然知之甚少，特别是 在多细胞生物中，有两个主要原因：（1）复制起始涉及瞬时蛋白质-蛋白质， 蛋白质-DNA相互作用是非常具有挑战性的研究，利用现有的生物化学，遗传学和结构 方法;（2）复制起始的最全面的研究已经在酵母中完成，但关键 复制起始的调节因子在酵母和后生动物之间保守性差。值得注意的是，一些人类 复制因子比它们的酵母对应物大2- 3倍，并且含有未知的新结构域， 功能协调发展的 我的实验室最近开发了一个单分子成像平台， 在真实的时间里。我们将使用这种强大的方法（i）提供一个定量的描述复制启动; (ii)了解这一过程如何从酵母到人类的变化;（iii）描述关键复制蛋白的作用 和他们的域是唯一的后生动物;和（iv）了解为什么突变在一些复制 引发因素导致疾病。我们的工作也将提供有价值的机制见解，癌细胞如何 通过过度激活复制起始来维持快速增殖，并告知复制起始因子如何 可用作新疗法的生物标志物或靶点。

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.