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 ORC 在哺乳动物细胞中的行为与其在单细胞真核生物（如酵母）中的行为显着不同。酵母 ORC 由六个不同亚基的稳定复合物组成，这些亚基在整个细胞分裂过程中始终与染色质结合，并靶向特定的 DNA 序列。相比之下，哺乳动物 ORC 由 Orc2 到 Orc5 的稳定核心复合物 ORC(2-5) 组成，与 Orc1 和 Orc6 相互作用较弱。然而，Orc1 与 ORC(2-5) 的结合对于复制前复合物组装和 DNA 复制至关重要。然而，在体外，后生动物 ORC 除了偏爱不对称的 A:T 丰富区域外，对特定 DNA 序列几乎没有亲和力。然而，在哺乳动物和果蝇的分化细胞中，ORC 位于与 DNA 复制起点一致的特定基因组位点。因此，ORC 激活特定复制起点的能力似乎取决于其与细胞核内存在的 DNA 相互作用的能力，这种相互作用似乎受到 Orc1 的调节。在过去的一年里，我们建立了 ORC 循环的基本特征，这是我们之前提出的一种限制 DNA 复制事件启动的调控途径，以便基因组在每次细胞分裂时复制一次且仅一次。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点的关联，并且是通过其 BAH 结构域实现的。该结构域与另一种尚未鉴定的蛋白质相互作用，当细胞退出有丝分裂并开始新的细胞分裂周期时，ORC 与染色体结合所需的蛋白质。此外，如果 Orc1 不与 ORC(2-5) 结合，那么它可以诱导细胞凋亡（程序性细胞死亡）。然而，如果未结合的 Orc1 是单泛素化的或磷酸化的（Orc1 的两种正常细胞周期依赖性修饰），则修饰的 Orc1 位于细胞质中，无法启动复制。这说明了 Orc1 被修饰，并且在一些细胞中随着细胞进入 S 期（DNA 复制期）而降解。此外，Orc1 的修饰形式不会诱导细胞凋亡。因此，细胞周期修饰调节 Orc1 活性，而 Orc1 活性调节 ORC 活性，ORC 活性调节 DNA 复制的起始。更多信息请访问我们的网站 (http://depamphilislab.nichd.nih.gov/)。规格 1) 真核生物中 DNA 复制起始位点的选择是由起点识别复合物 (ORC) 和基因组 DNA 之间的相互作用决定的。在哺乳动物细胞中，这种相互作用似乎受到 Orc1 的调节，Orc1 是唯一包含 BAH 结构域的 ORC 亚基。由于 BAH 结构域介导蛋白质-蛋白质相互作用，因此人类 Orc1 BAH 结构域发生突变，并且突变蛋白在人类细胞中表达，以确定它们对 ORC 功能的影响。 Orc1 的核定位、Orc1 与其他 ORC 亚基的关联或 Orc1 在 S 期的选择性降解不需要 BAH 结构域。然而，它确实促进了 Orc1 在 M 期到 G1 期转变期间与染色体的重新结合，并且是 Orc1 与 Epstein-Barr 病毒 oriP 结合并刺激 oriP 依赖性质粒 DNA 复制所必需的。此外，当细胞退出有丝分裂时，BAH 结构域影响 Orc1 促进 Orc2 与染色质结合的能力。因此，人 Orc1 中的 BAH 结构域通过促进 ORC 与染色质的结合来增强其在体内激活复制起点的能力。 2）先前的研究表明，哺乳动物起源识别复合物（ORC）的活性受到其Orc1亚基的细胞周期依赖性变化的调节。在这里，我们表明，Orc1 修饰（例如分别在 S 期和 G2/M 期正常发生的单泛素化和过度磷酸化）可以导致 Orc1 在细胞质中积累。这将抑制复制前复合物的重新组装，直到有丝分裂完成。在没有这些修饰的情况下，Orc1 的瞬时表达迅速诱导不依赖于 p53 的细胞凋亡，并且 Orc1 在核周而不是在整个细胞核中均匀积累。这种行为模仿了增殖细胞培养物中自发产生的凋亡细胞中内源性 Orc1 的浓度增加和核周积累。值得注意的是，在等量的 Orc2（唯一不诱导细胞凋亡的 ORC 亚基）存在的情况下，Orc1 的表达阻止了细胞凋亡的诱导并恢复了 Orc1 的均匀核定位。这将促进 ORC：染色质位点的组装，例如在 M 到 G1 相转变期间发生的情况。这些结果为支持 Orc1 的调节作用提供了直接证据，并表明哺乳动物发育过程中的异常 DNA 复制可能通过未修饰的 Orc1 的出现导致细胞凋亡。 3) 真核 DNA 复制始于六亚基起点识别复合物 (ORC) 与 DNA 的结合。为了研究哺乳动物 ORC 蛋白在其天然环境中的组装和功能，构建了 HeLa 细胞，该细胞持续表达经过基因改造的带有表位标记的重组人 Orc2 亚基。对这些细胞系的分析表明，Orc2 含有一个在体内与所有其他 ORC 亚基相互作用所需的单个 ORC 组装结构域，以及 ORC 在细胞核中积累所需的两个核定位信号 (NLS)。重组Orc2以ORC2-5或ORC1-5复合体的形式存在于细胞核中；没有检测到 ORC 亚基的其他组合。此外，只有 ORC1-5 与染色质部分结合，表明体内需要 Orc1 将 ORC2-5 加载到染色质上。令人惊讶的是，重组Orc2抑制了内源性Orc2的表达，揭示哺乳动物细胞限制了Orc2的细胞内水平，从而限制了细胞核中ORC2-5的量。由于这种抑制仅需要 ORC 组装和 NLS 结构域，因此这些结构域似乎构成了 Orc2 的功能结构域。 4) 我有幸担任一本书的编辑，其中 72 位 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。这本书标志着首次将基因组复制的基本过程与人类遗传性疾病联系起来。