Regulation of Chromosomal DNA Replication Dynamics in S cerevisiae

项目总结 组成真核生物基因组的超长dna分子被包裹、折叠和环路以 紧凑和组织DNA以进行其基本活动，包括基因表达、DNA复制和 DNA修复。染色体的空间结构是如何调控dna过程的，目前还不清楚。 了解，并且确定的涉及因素相对较少。DNA复制起始点受 对其活性的表观遗传调控导致S期间差异复制时间和起始点效率 相位。对复制源的这种级别控制有助于确保适当级别的源活动 基因组稳定性，然而，负责这一调节的分子机制(S)仍然不清楚。 我们发现萌芽中的酵母Fox蛋白，Forkhead 1(Fkh1)和Forkhead 2(Fkh2)是关键的调节因子 复制起始点启动时间，大多数早期起始点在酵母基因组中启动所必需的。FKH1和/或 Fkh2(Fkh1/2)在某些起始处与特定序列结合(称为Fkh激活的起始点)。最新研究 表明Fkh1/2的一个关键功能是招募Dbf4依赖的激酶(DDK)，这是 复制起点启动。该项目旨在完全理解细胞周期调节的结合 Fkh1/2具有起源，Fkh1/2与DDK相互作用。我们已经确定了一个潜在的二聚化基序 Fkh1/2是其在起源调控中所必需的功能，但在转录调控等其他功能中不是必需的。 我们将确定这个基序在起源调控中的功能。Fkh1建立了原点计时程序 G1期，这与Fkh1激活的起源于晚期复制核的重新定位有关 从外周到早期复制的核内环境。这些东西的确切意义 移动尚不清楚，但很可能代表了将转变为复制的起始簇的组装 工厂。因此，这一系统为阐明基本原理提供了一个新颖而有力的机会 染色体动力学机制，这是这项提议的一个主要目标。我们将使用基因 消除候选调节蛋白剖析相关分子事件的功能的方法 与起源动力学和复制启动有关。我们还将开发新的工具来更好地分析蛋白质- DNA相互作用。最后，我们将进行实验，剥离表观遗传层的起源调控，以 揭示基于序列的起源激发的潜在决定因素。我们将确定对基因组的影响 这些监管层面的稳定性。这些研究有很大的潜力揭示新的分子事件 DNA水平控制染色体及其基本遗传要素动态的DNA水平，如 复制源。

PROJECT SUMMARY The extremely long DNA molecules that comprise eukaryotic genomes are wrapped, folded, and looped to compact and organize the DNA for its essential activities, including gene expression, DNA replication, and DNA repair. Exactly how the spatial architecture of chromosomes regulates DNA processes is still poorly understood, and relatively few involved factors have been identified. DNA replication origins are subject to epigenetic regulation of their activity resulting in differential replication timings and origin efficiencies during S phase. This level of control over replication origins helps ensure an appropriate level of origin activity for genome stability, however, the molecular mechanism(s) responsible for this regulation have remained obscure. We discovered the budding yeast Fox proteins, Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as key regulators of replication origin initiation timing, required for most early origin firing across the yeast genome. Fkh1 and/or Fkh2 (Fkh1/2) bind to specific sequences at some origins (called Fkh-activated origins). Recent studies indicate that a key function of Fkh1/2 is to recruit Dbf4-dependent kinase (DDK), which is required for replication origin initiation. This project is geared toward fully understanding the cell cycle-regulated binding of Fkh1/2 with origins and Fkh1/2 interactions with DDK. We have identified a potential dimerization motif in Fkh1/2 that is required for its function in origin regulation but not in other functions like transcriptional regulation. We will determine the function of this motif in origin regulation. Fkh1 establishes the origin-timing program in G1 phase and this correlates with re-localization of Fkh1-activated origins from the late-replicating nuclear periphery to an early-replicating interior environment of the nucleus. The exact significance of these movements is unclear but likely represent the assembly of origin clusters that will transform into replication factories. Therefore, this system provides a novel and powerful opportunity to elucidate fundamental mechanisms of chromosomal dynamics, which is a major goal of this proposal. We will use genetic approaches to eliminate function of candidate regulator proteins to dissect the molecular events associated with origin dynamics and replication initiation. We will also develop new tools for better analysis of protein- DNA interactions. Finally, we will perform experiments to strip away epigenetic layers of origin regulation to reveal the underlying sequence-based determinants of origin firing. We will determine the impact on genome stability of these layers of regulation. These studies have strong potential to reveal novel molecular events at the DNA level governing the dynamics of chromosomes and their essential genetic elements, such as replication origins.