Initiation of DNA Replication in Mammalian Cells

哺乳动物细胞中 DNA 复制的启动

• 批准号: 10014364
• 负责人: mirit aladjem
• 金额: $ 163.9万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10014364
• 关键词: Affect; Antineoplastic Agents; Back; Binding; Binding Sites; Bioinformatics; Cancer cell line; Cell Cycle; Cell Cycle Checkpoint; Cells; Chemotherapy-Oncologic Procedure; Chromatin; Chromatin Loop; Chromosomes; Clinical; Communities; Complex; DNA; DNA Binding; DNA Repair Pathway; DNA Sequence; DNA biosynthesis; DNA sequencing; DNA-Protein Interaction; Data Set; Deacetylase; Defect; Developmental Therapeutics Program; Diploidy; Distal; Drug Targeting; Elements; Epigenetic Process; Eukaryotic Cell; Event; Exposure to; Family; Fiber; Frequencies; Future; Genetic Transcription; Genome; Genome Stability; Health; Human; Lead; Learning; Location; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Mitosis; Mitotic; Modification; Molecular; Mutation; Nature; Pathway interactions; Pharmaceutical Preparations; Phosphorylation Site; Play; Process; Proteins; Replication Initiation; Replication Origin; Replication-Associated Process; Role; SIRT1 gene; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Subgroup; Testing; Tissues; Transact; Translating; Work; anti-cancer; base; beta Globin; cancer cell; cancer therapy; cell growth; cell type; chromatin modification; genome-wide; insight; member; mutant; prevent; protein complex; recruit; repaired; replicator; response; tool; ubiquitin ligase

Within eukaryotic cells, genome duplication initiates at multiple sites on each chromosome. Replication initiation events in diploid mitotic cells proceed in a precise order and are strictly regulated by a series of cell cycle checkpoint signaling pathways. These regulatory constraints, however, are often relaxed in cancer cells. Because the processes that coordinate replication ultimately converge on chromatin, understanding the molecular events that precede DNA replication at the chromatin level is crucial if we are to fully understand cell growth. Critical information about this process is missing because protein complexes that initiate chromosomal replication seem to bind DNA indiscriminately. To gain a complete understanding of the DNA replication process we must resolve how this non-specific DNA binding translates into highly coordinated replication. Our studies are based on the hypothesis that sequence-specific signaling molecules associate with replication initiation sites on chromatin where they modulate the local activity of the ubiquitous replication machinery and dictate both the location and timing of replication initiation events. To test this hypothesis, we characterize protein-DNA interactions at replication initiation sites and identify interactions that play regulatory roles in the DNA replication process. We started by identifying distinct DNA sequences, termed replicators, which facilitate the initiation of DNA replication. We have initially identified these replicator sequences and we now use them as bait to isolate protein complexes that potentially regulate replication. In recent studies we have identified two discrete DNA-protein complexes within one replicator element. One of these complexes includes RepID, a member of the DDB1-Cul4-associated-factor (DCAF) family, which binds a subset of replication initiation sites and is required for replication at those sites. We found that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. Our recent studies show that RepID exerts its effects on replication by recruiting a ubiquitin ligase complex, CRL4, to chromatin, suggesting that ubiquitin ligase complexes play a role in regulating DNA replication dynamics. Importantly, RepID binding origins require RepID for initiation of DNA replication, providing the first example of a site-specific interaction that determines the initiation of DNA replication on a group of metazoan replication origins. Current studies characterize another role of RepID and CRL4 in insuring proper progression during mitosis. In other studies we have shown that replication origins bind another protein, a phosphorylated form of the NAD+-dependent deacetylase SIRT1. Unlike RepID, SIRT1 is not required for initiation of DNA replication, and instead, it prevents replication from initiating in a subgroup of potential origins ("dormant origins"). In concordance, dormant replication origins are activated, and the overall frequency of replication initiation events increases, in cells that do not contain the phosphorylated form of SIRT1 (either due to a depletion or to a mutation in the phosphorylation site). In recent studies we have observed an increased frequency of replication initiation events in cells that contain a SIRT1 mutant in which deacetylase activity is inactivated, suggesting that suppression of dormant origins requires SIRT1's deacetylase activity. Cells with activated dormant origins harbor extrachromosomal elements and DNA breaks, suggesting that maintenance of origin dormancy by SIRT1 facilitates genomic stability. We are currently investigating how SIRT1 modulates replication origin activation in cells exposed to stressful conditions. Our studies are facilitated by tools we have developed to map replication initiation sites throughout the genome, and using these maps to analyze DNA replication in the context of chromatin modifications and transcriptional activity. Using a combination of DNA sequencing and single fiber analyses, have generated a comprehensive dataset of replication initiation sites for several human cancer cell lines. We have demonstrated that replication origin usage varies with tissue type, with distinct modifications associated with cell-type specific replication origins. To facilitate these studies, we are continuously developing bioifnromatics tools to help decipher the relationships among RepID binding sites and epigenetic features. We are making those tools available to the community to support bioinformatics characterization of DNA-protein interaction loci. An important aspect of our work pertinent to human health is the response of the replication machinery to perturbations. A large and increasing number of anti-cancer drugs target DNA replication or interfere with cell cycle signaling. Understanding specific cell cycle defects in different cancers is likely to provide clues regarding their sensitivity to anti-cancer therapies. We are currently studying replication origins activated in response to those drugs, directly mapping chromatin targets involved in preventing excess replication. Our strategy consists of combining genome-scale sequencing with single-fiber analyses. This approach can provide important insights into the organization of replication initiation events and the cellular responses to signals that might perturb DNA replication. We ask how particular replication and repair pathways affect the pace and frequency of DNA replication. We are currently involved in several collaborative studies characterizing replication dynamics following exposure to anti-cancer chemotherapy. In the future, we will investigate how protein-DNA interactions that are required for DNA replication are modulated in response to environmental challenges and anti-cancer drugs. As we learn more about local and distal interactions that promote DNA replication, we will continue to explore pathways that signal back from chromatin to the cell cycle machinery to affect the replication landscape and modulate the response to anti-cancer therapy.

在真核细胞中，基因组复制始于每条染色体上的多个位点。二倍体有丝分裂细胞的复制起始事件按照精确的顺序进行，并受到一系列细胞周期检查点信号通路的严格调控。然而，这些调控约束在癌细胞中通常是放松的。因为协调复制的过程最终集中在染色质上，所以如果我们要充分了解细胞生长，了解染色质水平上DNA复制之前的分子事件是至关重要的。由于启动染色体复制的蛋白质复合物似乎不加选择地结合DNA，因此关于这一过程的关键信息缺失。为了全面了解DNA复制过程，我们必须解决这种非特异性DNA结合如何转化为高度协调的复制。我们的研究基于这样的假设，即序列特异性信号分子与染色质上的复制起始位点相关，在染色质上，它们调节无处不在的复制机制的局部活性，并决定复制起始事件的位置和时间。为了验证这一假设，我们表征了复制起始位点的蛋白质-DNA相互作用，并确定了在DNA复制过程中发挥调节作用的相互作用。我们首先识别不同的DNA序列，称为复制子，它促进DNA复制的开始。我们已经初步确定了这些复制子序列，现在我们用它们作为诱饵来分离可能调节复制的蛋白质复合物。在最近的研究中，我们在一个复制因子元件中确定了两个离散的dna -蛋白质复合物。这些复合体之一包括RepID，它是ddb1 - cul4相关因子（DCAF）家族的成员，它结合复制起始位点的一个子集，并且是在这些位点进行复制所必需的。我们发现，RepID与复制子元件与人类β -珠蛋白（HBB）位点内远端调控序列之间的染色质环相互作用有关。我们最近的研究表明，RepID通过向染色质募集一个泛素连接酶复合物CRL4来发挥其对复制的影响，这表明泛素连接酶复合物在调节DNA复制动力学中起作用。重要的是，RepID结合起点需要RepID来启动DNA复制，这提供了第一个位点特异性相互作用的例子，该相互作用决定了一组后生动物复制起点上DNA复制的启动。目前的研究表明，在有丝分裂过程中，RepID和CRL4在确保正常进展中的另一个作用。在其他研究中，我们已经证明复制起点结合另一种蛋白质，一种NAD+依赖性脱乙酰酶SIRT1的磷酸化形式。与RepID不同的是，SIRT1不是DNA复制起始所必需的，相反，它可以防止复制在潜在起始点（“休眠起始点”）的子组中开始。与此一致的是，在不含SIRT1磷酸化形式的细胞中（由于磷酸化位点的耗尽或突变），休眠复制起点被激活，复制起始事件的总体频率增加。在最近的研究中，我们观察到在含有去乙酰化酶活性失活的SIRT1突变体的细胞中，复制起始事件的频率增加，这表明抑制休眠起源需要SIRT1的去乙酰化酶活性。激活休眠起源的细胞含有染色体外元件和DNA断裂，表明SIRT1维持起源休眠有助于基因组稳定性。我们目前正在研究SIRT1如何调节应激条件下细胞的复制起始激活。我们的研究是通过我们开发的工具来绘制整个基因组的复制起始位点，并使用这些地图来分析染色质修饰和转录活性背景下的DNA复制。利用DNA测序和单纤维分析的结合，已经生成了几种人类癌细胞系复制起始位点的综合数据集。我们已经证明，复制起始点的使用随组织类型的不同而不同，与细胞类型特异性复制起始点相关的修饰也不同。为了促进这些研究，我们正在不断开发生物信息学工具，以帮助破译RepID结合位点和表观遗传特征之间的关系。我们正在将这些工具提供给社区，以支持dna -蛋白质相互作用位点的生物信息学表征。我们与人类健康有关的工作的一个重要方面是复制机制对扰动的反应。越来越多的抗癌药物靶向DNA复制或干扰细胞周期信号。了解不同癌症中特定的细胞周期缺陷可能为它们对抗癌治疗的敏感性提供线索。我们目前正在研究这些药物激活的复制起点，直接绘制染色质目标，以防止过度复制。我们的策略包括将基因组规模测序与单纤维分析相结合。这种方法可以为研究复制起始事件的组织和细胞对可能干扰DNA复制的信号的反应提供重要的见解。我们询问特定的复制和修复途径如何影响DNA复制的速度和频率。我们目前参与了几个合作研究，描述了暴露于抗癌化疗后的复制动力学。在未来，我们将研究如何调节DNA复制所需的蛋白质-DNA相互作用，以应对环境挑战和抗癌药物。随着我们更多地了解促进DNA复制的局部和远端相互作用，我们将继续探索从染色质到细胞周期机制的信号返回途径，以影响复制景观并调节对抗癌治疗的反应。