DNA Replication-Linked Chromatin Assembly in Yeast

真核细胞染色体中的DNA与组蛋白结合。组蛋白的八聚体被组织成核小体，核小体是染色质的基本构件。核小体使DNA紧凑，并调节基因组对DNA新陈代谢的各个方面的可及性。在DNA复制过程中，组蛋白被快速组装到子体DNA链上；在没有组蛋白的情况下进行DNA复制会导致致命性。这种有序的沉积是由蛋白质介导的，这些蛋白质结合新合成的组蛋白并将它们沉积到DNA上。核小体组装蛋白包括染色质组装因子-I和Asflp，它们广泛存在于真核生物中。CAF-I和Asflp在体外协同作用形成核小体；在体内，它们在特定区域建立染色质，例如抑制基因转录的异染色质基因。CaF-I对着丝粒的染色质结构也很重要。这项工作的长期目标是了解Caf-I、Asflp和其他组蛋白沉积蛋白是如何协同发挥作用的，以及这些蛋白是如何在不同的位点上进行差异调控的。我们在萌芽酵母酿酒酵母中研究了这些问题，酿酒酵母是一种生物化学和遗传上易驯化的生物。我们的具体目标包括：1.开发研究特定生化反应中组蛋白沉积的试剂。我们将在这个系统中测试突变体，以在机制水平上了解我们观察到的沉默表型。该系统还将使发现刺激或调节组蛋白沉积的新蛋白质成为可能。2.组蛋白沉积蛋白在着丝粒中的特殊作用。我们将确定着丝粒的哪些方面。我们将确定着丝粒染色质的哪些方面是由组蛋白沉积蛋白构建的，并研究这些蛋白质是如何被招募到着丝粒的。3.不同组蛋白沉积蛋白细胞分布的测定。我们还将探索参与DNA损伤修复和基因沉默的蛋白质如何在不同的位点以不同的方式调节组蛋白的沉积。对这些高度保守的组蛋白沉积蛋白的机械理解将适用于所有真核生物，包括人类。同样，发现感受DNA损伤的蛋白质如何调节核小体形成将提高我们对后生动物中的突变和癌症的理解。染色体的正确组装和功能对细胞周期进程和基因表达也很重要。因此，本建议中所研究的人类蛋白质同源物是开发新的抗增殖药物的良好候选靶点。

The DNA in eukaryotic chromosomes is bound by histone proteins. Octamers of histone proteins are organized into nucleosomes, the fundamental building blocks of chromatin. Nucleosomes compact the DNA and regulate the accessibility of the genome to all aspects of DNA metabolism. During DNA replication, histones are rapidly assembled onto daughter DNA strands; DNA replication in the absence of histones results in lethality. This ordered deposition is mediated by proteins that bind newly synthesized histones and deposit them onto DNA. Nucleosome assembly proteins include Chromatin Assembly Factor-I (CAF-I) and Asflp, which are ubiquitous among eukaryotic organisms. CAF-I and Asflp act synergistically in vitro to form nucleosomes; in vivo, they build chromatin at specialized regions, such as heterochromatic loci that silence gene transcription. CAF-I is also important for chromatin structure at centromeres. The long-term goals of this work are to understand how CAF-I, Asflp, and other histone deposition proteins function synergically, and how these proteins are differentially regulated at different loci. We study these questions in the budding yeast Saccharomyces cerevisiae, a biochemically and genetically tractable organism. Our specific aims include: 1. Development of reagents to study histone deposition in a defined biochemical reaction. We will test mutant in this system to understand at a mechanistic level silencing phenotypes we have observed. This system will also enable discovery of new proteins that stimulate or regulate histone deposition. 2. Investigation of the special role of histone deposition proteins at centromeres. We will determine which aspects of centromeres. We will determine which aspects of centomeric chromatin are built by histone deposition proteins, and investigate how these are recruited to centromeres. 3. Determination of the cellular distribution of the different histone deposition proteins. We will also explore how proteins involved in DNA damage repair and gene silencing regulate histone deposition differently at different loci. Mechanistic understanding of these highly conserved histone deposition proteins will be applicable to all eukaryotic organisms, including humans. Likewise, discovery of how proteins that sense DNA damage regulate nucleosome formation will improve our understanding of mutagenesis and cancer in metazoans. Proper assembly and function of chromosomes is also important for cell cycle progression and gene expression. Therefore, human homologs of proteins investigated in this proposal are good candidate targets for developing new anti- proliferative drugs.