DNA Replication-Linked Chromatin Assembly in Yeast

真核生物染色体中的DNA由组蛋白结合。组蛋白的八聚体组成核小体，核小体是染色质的基本组成部分。核小体压缩DNA并调节基因组对DNA代谢各方面的可及性。在DNA复制过程中，组蛋白迅速组装到子DNA链上；没有组蛋白的DNA复制会导致死亡。这种有序的沉积是由结合新合成的组蛋白并将其沉积到DNA上的蛋白质介导的。核小体组装蛋白包括染色质组装因子- 1 （cafi）和Asflp，它们普遍存在于真核生物中。ca - 1和Asflp在体外协同作用形成核小体；在体内，它们在特定区域构建染色质，如异染色质位点，使基因转录沉默。caf1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ca - 1， Asflp和其他组蛋白沉积蛋白如何协同作用，以及这些蛋白如何在不同位点受到差异调节。我们研究了这些问题的出芽酵母酿酒酵母，一种生物化学和遗传上易于处理的生物。我们的具体目标包括：1。开发用于研究组蛋白沉积在特定生化反应中的试剂。我们将在这个系统中测试突变体，以了解我们观察到的沉默表型的机制水平。该系统还将有助于发现刺激或调节组蛋白沉积的新蛋白质。2. 组蛋白沉积蛋白在着丝粒中的特殊作用的研究。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，并研究这些方面如何被招募到着丝粒中。3. 测定不同组蛋白沉积蛋白的细胞分布。我们还将探讨参与DNA损伤修复和基因沉默的蛋白质如何在不同的位点上以不同的方式调节组蛋白沉积。对这些高度保守的组蛋白沉积蛋白的机制理解将适用于包括人类在内的所有真核生物。同样，发现感知DNA损伤的蛋白质如何调节核小体的形成将提高我们对后生动物的突变和癌症的理解。染色体的正常组装和功能对细胞周期的进展和基因的表达也很重要。因此，本文研究的人类同源蛋白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.