Initiation Of DNA Replication In Mammalian Chromosomes

哺乳动物染色体中 DNA 复制的起始

基本信息

批准号：
7594135
负责人：
Melvin DePamphilis
金额：
$ 95.98万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7594135
关键词：
Accounting Adult Affect Affinity Age Animals Apoptosis Apoptotic Appearance Behavior Binding Binding Sites Books Cell Communication Cell Cycle Cell Line Cell Nucleus Cell division Cells Chromatin Chromatin Structure Chromosomes Cleaved cell Complex Cultured Cells Cytoplasm DNA DNA Methylation DNA Sequence DNA Virus Infections DNA biosynthesis DNA replication origin Deoxyribonucleotides Development Disease Embryo Environment Epigenetic Process Epitopes Eukaryota Eukaryotic Cell Event Exhibits Fishes G1 Phase Generations Genes Genetic Genetic Transcription Genome Genomic Instability Genomics Goals Hela Cells Human Human Herpesvirus 4 In Vitro Induction of Apoptosis Inherited Internet Laboratories Life Link Localized Location Malignant Neoplasms Mammalian Cell Mammalian Chromosomes Mammals Mediating Mitosis Modeling Modification Mono-S Monoubiquitination Mutate Mutation Normal Cell Nuclear Nuclear Localization Signal Numbers Organism Phase Phase Transition Process Proliferating Protein Binding Proteins Rana Recombinants Regulatory Pathway Replication Initiation Replication Origin Replication-Associated Process Role Site Staging Structure Time Viral Yeasts accomplished suicide concept density fly gene repair human disease in vivo initiation site of DNA replication mutant origin recognition complex plasmid DNA preference prevent programs protein protein interaction transcription factor

项目摘要

Several years ago, we discovered that the behavior of ORC in mammalian cells differs significantly from its behavior in single cell eukaryotes such as yeast. Yeast ORCs consist of a stable complex of six different subunits that remain bound to chromatin throughout cell division and target specific DNA sequences. In contrast, mammalian ORCs consists of a stable core complex ORC(2-5) of Orc2 through Orc5 that interacts weakly with Orc1 and Orc6. Nevertheless, the association of Orc1 with ORC(2-5) is essential for prereplication complex assembly and DNA replication. In vitro, however, metazoan ORCs exhibit little affinity for specific DNA sequences other than a preference for asymmetric A:T-rich regions. Nevertheless, in the differentiated cells of mammals and flies, ORCs is localized at specific genomic sites that are coincident with DNA replication origins. Thus, the ability of ORC to activate a particular replication origin appears to depend on its ability to interact with DNA as it exists within the nucleus, an interaction that appears to be regulated by Orc1. During the past year, we established the basic features of the ORC cycle, a regulatory pathway we proposed previously that restricts initiation of DNA replication events so that genomes are duplicated once and only once each time a cell divides. The largest subunit (Orc1) regulates association of the stable ORC(2-5) core complex with replication origins in vivo, and that it does so through its BAH domain. This domain interacts with another, as yet unidentified, protein that is required for ORC binding to chromosomes as cells exit mitosis and begin a new cell division cycle. Moreover, if Orc1 is not bound to ORC(2-5), then it can induce apoptosis (programmed cell death). However, if the unbound Orc1 is either mono-ubiquitinated or phosphorylated (two normal cell cycle dependent modifications of Orc1), then the modified Orc1 is localized in the cytoplasm where it cannot initiate replication. This accounts for the fact that Orc1 is modified and in some cells degraded as the cells enter S-phase (period of DNA replication). In addition, the modified forms of Orc1 do not induce apoptosis. Thus, cell cycle modifications regulate Orc1 activity, and Orc1 activity regulates ORC activity, which regulates initiation of DNA replication. Further information is found at our web site (http://depamphilislab.nichd.nih.gov/). Specifics 1) Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a BAH domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1s ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin. 2) Previous studies have suggested that the activity of the mammalian origin recognition complex (ORC) is regulated by cell cycle dependent changes in its Orc1 subunit. Here we show that Orc1 modifications such as monoubiquitination and hyperphosphorylation that occur normally during S and G2/M phases, respectively, can cause Orc1 to accumulate in the cytoplasm. This would suppress reassembly of prereplication complexes until mitosis is complete. In the absence of these modifications, transient expression of Orc1 rapidly induced p53-independent apoptosis, and Orc1 accumulated perinuclearly rather than uniformly throughout the nucleus. This behavior mimicked the increased concentration and perinuclear accumulation of endogenous Orc1 in apoptotic cells that arise spontaneously in proliferating cell cultures. Remarkably, expression of Orc1 in the presence of an equivalent amount of Orc2, the only ORC subunit that did not induce apoptosis, prevented induction of apoptosis and restored uniform nuclear localization of Orc1. This would promote assembly of ORC:chromatin sites, such as occurs during the M to G1-phase transition. These results provide direct evidence in support of the regulatory role proposed for Orc1, and suggest that aberrant DNA replication during mammalian development could result in apoptosis through the appearance of unmodified Orc1. 3) Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC2-5 or ORC1-5 complex; no other combinations of ORC subunits were detected. Moreover, only ORC1-5 was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC2-5 onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC2-5 in the nucleus. Since this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2. 4) I was privleged to act as editor for a book in which 72 leaders in the field of DNA replication described the various aspects of cellular and viral DNA replication and their relationships to human disease. This book marks a the first such effort to link the fundamental process of genome duplication to genetically inherited diseases in humans.

几年前，我们发现兽人在哺乳动物细胞中的行为与在酵母等单细胞真核生物中的行为显着不同。酵母菌由六个不同亚基的稳定复合物组成，这些复合物在整个细胞分裂和靶向特定的DNA序列中仍然与染色质结合。相比之下，哺乳动物兽人由ORC5的稳定核心复合物ORC（2-5）与ORC5与ORC1和ORC6微弱相互作用组成。然而，ORC1与ORC（2-5）的关联对于预先复杂的组装和DNA复制至关重要。然而，在体外，后生兽人对特定的DNA序列的亲和力几乎没有，而不是偏爱非对称A：富含T的区域。然而，在哺乳动物和果蝇的分化细胞中，兽人位于与DNA复制起源一致的特定基因组部位。因此，ORC激活特定复制起源的能力似乎取决于其与DNA相互作用的能力，因为它存在于核内，这种相互作用似乎受ORC1调节。在过去的一年中，我们建立了ORC循环的基本特征，ORC周期是一种调节途径，我们先前提出的限制了DNA复制事件的启动，以便每次分裂一次基因组一次复制一次。最大的亚基（ORC1）调节稳定的ORC（2-5）核心复合物与体内复制起源的关联，并且它通过其BAH域进行。该结构域与另一种尚未确定的蛋白质相互作用，这是兽人与染色体结合时所需的蛋白质，因为细胞出口有丝分裂并开始新的细胞分裂周期。此外，如果ORC1与兽人（2-5）无约束，则可以诱导凋亡（编程细胞死亡）。但是，如果未结合的ORC1是单泛素化的或磷酸化的（两个正常的细胞周期依赖性ORC1的修饰），则修饰的ORC1位于细胞质中，无法引发复制。这说明了ORC1经过修改并在某些细胞进入S期（DNA复制周期）时降解的事实。另外，ORC1的修饰形式不会诱导凋亡。因此，细胞周期修饰调节ORC1活性，ORC1活性调节ORC活性，从而调节DNA复制的起始。在我们的网站（http://depamphilislab.nichd.nih.gov/）上找到更多信息。细节 1）选择真核生物中DNA复制的起始位点的选择取决于原点识别复合物（ORC）和基因组DNA之间的相互作用。在哺乳动物细胞中，这种相互作用似乎受ORC1的调节，ORC1是唯一包含BAH结构域的ORC亚基。由于BAH结构域介导了蛋白质 - 蛋白质相互作用，因此人类ORC1 BAH结构域被突变，突变蛋白在人类细胞中表达以确定其对ORC功能的影响。 ORC1的核定位，ORC1与其他ORC亚基的关联或S相期间ORC1的选择性降解不需要BAH域。但是，它确实促进了在M到G1期间转变期间ORC1与染色体的重新关联，并且与Epstein-Barr病毒Orip结合ORIP和刺激依赖Orip依赖性的质粒DNA的复制是必需的。此外，BAH结构域影响了ORC1促进ORC2与染色质结合的能力，因为细胞出口有丝分裂。因此，人ORC1中的BAH结构域通过促进ORC与染色质的关联来促进其在体内激活复制起源的能力。 2）先前的研究表明，哺乳动物起源识别复合物（ORC）的活性受其ORC1亚基的细胞周期依赖性变化调节。在这里，我们表明，通常在S和G2/M相通常发生的ORC1修饰，例如单次泛素化和过度磷酸化，可能会导致ORC1在细胞质中积累。这将抑制预先复合络合物的重新组装，直到有丝分裂完成为止。在没有这些修饰的情况下，ORC1的瞬时表达迅速诱导了p53非依赖性凋亡，而ORC1在整个核中累积了perinucice，而不是均匀地累积。这种行为模仿了内源性ORC1在凋亡细胞中内源性ORC1的浓度增加，而在增殖细胞培养物中会自发产生。值得注意的是，ORC1在存在等效量的ORC2的情况下的表达，ORC2是唯一没有诱导凋亡的ORC亚基，可以阻止凋亡诱导并恢复了ORC1的均匀核定位。这将促进ORC的组装：染色质位点，例如在M到G1相过渡期间发生的。这些结果提供了直接的证据，以支持ORC1提出的调节作用，并表明在哺乳动物发育过程中的异常DNA复制可能通过出现未修饰的ORC1而导致凋亡。 3）真核DNA复制始于六个亚基起源识别复合物（ORC）与DNA的结合。为了研究哺乳动物兽人在其天然环境中的组装和功能，构造了HeLa细胞，该细胞构成了组成型表达的表位，标记为遗传改变的重组人ORC2亚基。对这些细胞系的分析表明，ORC2包含一个单个ORC组装结构域，该结构域与所有其他ORC亚基相互作用以及两个核定位信号（NLSS）所需的单个ORC组装结构域（NLSS）所需的单个ORC组装结构域，这些信号（NLSS）是兽人积聚在核中所需的。重组ORC2在细胞核中以ORC2-5或ORC1-5复合物而存在。未检测到兽人亚基的其他组合。此外，仅ORC1-5与染色质分数结合，这表明在体内需要ORC1将ORC2-5加载到染色质上。出乎意料的是，重组ORC2抑制了内源性ORC2的表达，揭示了哺乳动物细胞限制了细胞内ORC2的水平，从而限制了核中ORC2-5的量。由于这种抑制仅需要ORC组件和NLS域，因此这些域似乎构成了ORC2的功能域。 4）我很愿意担任一本书的编辑，该书中有72名DNA复制领域的领导者描述了细胞和病毒DNA复制的各个方面及其与人类疾病的关系。这本书标志着将基因组重复的基本过程与人类遗传遗传疾病联系起来的第一个努力。