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Initiation of DNA Replication in Mammalian Cells

哺乳动物细胞中 DNA 复制的启动

基本信息

批准号：
10926012
负责人：
mirit aladjem
金额：
$ 190.94万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
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/10926012
关键词：
Address Affect Anaphase Back Binding Biochemical Bioinformatics Cell Cycle Cell Proliferation Cell division Cells Chromatin Chromosome abnormality Chromosomes Clinical Collaborations Complex Consensus DNA DNA Damage DNA Repair Pathway DNA Sequence DNA biosynthesis DNA replication fork DNA-Protein Interaction Development Developmental Therapeutics Program Distal Elements Energy Metabolism Ensure Epigenetic Process Event Exhibits Exposure to Fiber Future Genetic Genome Genome Stability Genomic Instability Health Human Image Learning Link Location Malignant Neoplasms Mammalian Cell Maps Mediating Metabolic Metaphase Mitosis Molecular Molecular Machines Nature Oncogenes Pathway interactions Phosphotransferases Play Property Protein Family Proteins Regulation Replication Initiation Replication Origin Replication-Associated Process Research Personnel Role SIRT1 gene Set protein Signal Pathway Signal Transduction Site Specificity TOPBP1 Gene Techniques Testing Translating Work anti-cancer therapeutic biochemical tools cancer cell cancer therapy cell growth cell type chemotherapeutic agent chromatin modification chromosome replication cis acting element computerized tools genome integrity genome-wide histone modification inhibitor insight member novel prevent programs protein complex protein protein interaction recruit replication stress response segregation tool translational potential treatment response tumorigenesis ubiquitin ligase whole genome

项目摘要

Our studies focus on cellular signaling pathways that regulate the location, timing and progression of DNA synthesis. We have identified cis-acting elements that facilitate the initiation of DNA replication, generated whole-genome-scale maps of replication initiation sites in human cells and detected novel protein-DNA interactions at replication initiation sites (replication origins). These discoveries were enabled by novel bioinformatics and biochemical approaches that we developed and implemented. Mapping replication origin activity and characterizing replication fork progression have demonstrated strong links between replication, histone modifications and chromatin packaging. The observed highly orchestrated order of the DNA replication program, however, contrasts with the low sequence-specificity exhibited by the molecular machines that catalyze DNA synthesis and the absence of a "consensus" DNA sequence that identifies all replication origins. We address this challenge by proposing and testing the hypothesis that genome duplication is guided by nuanced, selective protein-DNA interactions at discrete groups of replication origins that share distinct features. In the recent review period, we used a combination of genetic, biochemical, bioinformatics, imaging and functional analyses to study DNA-protein interactions and chromatin transactions that govern the initiation of DNA replication. We identified protein complexes that selectively assemble on groups of replication origins and modify their initiation capacity, providing the first example of site-specific interactions that modulate the initiation of DNA replication. The approach exemplified in our studies can pave a path towards a complete understanding of the interactions that spatially and temporally orchestrate chromosome duplication. Our current studies focus on two sets of protein-DNA interactions at replication origins. First, we found that the RepID protein, a member of the DDB1-Cul4-associated-factor (DCAF) protein family, preferentially interacts with replication origins. We have shown that RepID is required for initiation of DNA replication at the origins that bind it. We have further shown that RepID controls the replication program by recruiting the ubiquitin ligase complex, CRL4, to chromatin. In turn, RepID-recruited CRL4 prevents aberrant chromosome re-replication, ensuring that genome duplication occurs only once per cell division. We also discovered another function of RepID and CRL4 in regulating the metaphase-anaphase transition during mitosis. These observations have provided new insights into the mechanisms by which the fidelity of chromosome duplication and segregation can be compromised in cancer cells. Our current studies probe into the question of how cells orchestrate the activity of RepID and CRL4 with other ubiquitin ligases on chromatin to regulate cell proliferation, and characterize in detail the consequences of dysregulation of CRL4 chromatin recruitment and activity at replication origins. Given the mounting evidence that chromosomal re-replication and mis-segregation can be triggered by oncogenes at the onset of tumorigenesis, and the recent development of CRL4 inhibitors (e.g. NEDDylation inhibitors) as anti-cancer therapeutics, these studies have potential translational implications. Second, we identified an interaction between replication origins and the NAD+-dependent protein deacylase SIRT1. Unlike RepID, SIRT1 is not required for DNA replication, but instead, restricts the initiation of DNA replication to a particular group of origins ("baseline" origins) while preventing replication from initiating at other ("dormant") origins. This observation points to a mechanistic link between cellular energy metabolism, epigenetic marks and the regulation of replication origin activation, which play critical roles in maintaining genome integrity. Using SIRT1 activity as a molecular switch to turn dormant origins on and off, we have begun to characterize origin dormancy in detail, mapping the locations of dormant origins and identifying chromatin modifications that distinguish dormant from baseline origins. This work led us to identify components of a signaling network, involving the ATR kinase and the replication accessory protein TOPBP1, that relieves SIRT1-mediated origin dormancy when the baseline origins are stalled, as it occurs when cells are under replication stress. Pointing to the critical role in SIRT1 in maintaining origin dormancy and genome stability, cells with activated dormant origins harbor extrachromosomal elements and exhibit DNA breaks. Our current studies are focused on proteins that associate with dormant origins, the mechanism(s) by which SIRT1 suppresses initiation. We also study molecular pathways, including the abovementioned ATR pathway, that counter SIRT1-mediated suppression to activate dormant origins in cells exposed to stressful conditions. Because ATR inhibitors are explored, at the DTB and elsewhere, as promising therapy agents, these analyses also have a translational potential. The responses of the replication machinery to perturbations are pertinent to human health and the specific cell-cycle regulatory deficiencies in distinct cancer cell types are likely to provide clues to their sensitivity to therapy. Our studies demonstrate that deregulation of the early stages of DNA replication leads to excess replication and subsequent genomic instability through two distinct paths: one that involves activation of dormant origins, and another involving over-activation of baseline origins. We are currently characterizing genetic and epigenetic properties of the replication origins activated by each pathway, as well as protein-DNA interactions modulated by each pathway. Using a combination of single-fiber analyses and sequencing-based techniques, we analyze replication dynamics following exposure to chemotherapeutic agents and chromatin modulators. These studies are expected to identify chromatin targets that are normally involved in preventing excess replication and uncover signaling pathways that convey metabolic status to chromatin. As we learn more about local and distal interactions that promote DNA replication, we will explore pathways that signal back from chromatin to the cell cycle machinery to affect the replication landscape and the cellular responses to anticancer therapy. Our studies rely on tools we have developed to map replication initiation sites throughout the genome and compare replication initiation sites with distinct chromatin features. We are also collaborating with other investigators, within and outside NCI, to characterize genetic and epigenetic features of cancer cells and participate in collaborative efforts that link genetic and epigenetic signatures with responses to therapy. In future studies, we plan to further dissect the molecular interactions that regulate chromosome duplication. Specifically, using a combination of single-fiber analyses, biochemical and computational tools, we will systematically characterize protein-DNA and protein-protein interactions that mediate the effects of RepID, SIRT1, and DNA damage signaling pathways on the chromosome replication program.

我们的研究集中在调控DNA合成的位置、时间和进程的细胞信号通路上。我们已经确定了促进DNA复制起始的顺式作用元件，生成了人类细胞中复制起始位点的全基因组图谱，并在复制起始位点（复制起始点）检测到新的蛋白质-DNA相互作用。这些发现是由我们开发和实施的新型生物信息学和生化方法实现的。绘制复制起源活性和表征复制叉进展已经证明了复制、组蛋白修饰和染色质包装之间的紧密联系。然而，观察到的DNA复制程序的高度协调顺序与催化DNA合成的分子机器所表现出的低序列特异性和缺乏识别所有复制起源的“共识”DNA序列形成鲜明对比。我们通过提出和测试假设来解决这一挑战，即基因组复制是由细微的、选择性的蛋白质- dna相互作用指导的，这些相互作用在复制起源的离散组中具有不同的特征。在最近的回顾期间，我们使用遗传，生化，生物信息学，成像和功能分析的组合来研究DNA-蛋白质相互作用和染色质交易控制DNA复制的起始。我们发现了选择性组装在复制起点组上并修改其起始能力的蛋白质复合物，提供了第一个调节DNA复制起始的位点特异性相互作用的例子。在我们的研究中举例说明的方法可以为完全理解在空间和时间上协调染色体复制的相互作用铺平道路。我们目前的研究主要集中在复制起点的两组蛋白质- dna相互作用。首先，我们发现作为ddb1 - cul4相关因子（DCAF）蛋白家族成员的RepID蛋白优先与复制起点相互作用。我们已经证明，在结合它的起点上，启动DNA复制需要RepID。我们进一步证明，RepID通过将泛素连接酶复合物CRL4招募到染色质上来控制复制程序。反过来，repid募集的CRL4阻止异常的染色体再复制，确保每次细胞分裂只发生一次基因组复制。我们还发现了在有丝分裂过程中，RepID和CRL4调控中期-后期转变的另一个功能。这些观察结果为染色体复制和分离的保真度在癌细胞中受损的机制提供了新的见解。我们目前的研究探讨了细胞如何协调RepID和CRL4与其他泛素连接酶在染色质上的活性来调节细胞增殖，并详细描述了CRL4染色质募集和复制起点活性失调的后果。鉴于越来越多的证据表明，在肿瘤发生时，癌基因可以触发染色体再复制和错误分离，以及最近CRL4抑制剂（如neddyylation抑制剂）作为抗癌治疗药物的发展，这些研究具有潜在的翻译意义。其次，我们确定了复制起点与NAD+依赖性蛋白去乙酰化酶SIRT1之间的相互作用。与RepID不同，SIRT1不是DNA复制所必需的，相反，它将DNA复制的起始限制在特定的起始点组（“基线”起始点），同时阻止复制在其他（“休眠”起始点）起始。这一观察结果表明，细胞能量代谢、表观遗传标记和复制起始激活调控之间存在机制联系，它们在维持基因组完整性方面发挥着关键作用。利用SIRT1活性作为开启和关闭休眠起源的分子开关，我们已经开始详细描述起源休眠，绘制休眠起源的位置，并识别区分休眠起源和基线起源的染色质修饰。这项工作使我们确定了信号网络的组成部分，包括ATR激酶和复制辅助蛋白TOPBP1，当基线起源停滞时，sirt1介导的起源休眠会缓解，因为当细胞处于复制压力下时就会发生这种情况。SIRT1在维持起始点休眠和基因组稳定性方面发挥着关键作用，激活休眠起始点的细胞携带染色体外元件并表现出DNA断裂。我们目前的研究主要集中在与休眠起源相关的蛋白质，以及SIRT1抑制起始的机制。我们还研究了分子途径，包括上述的ATR途径，对抗sirt1介导的抑制，激活应激条件下细胞的休眠起源。由于ATR抑制剂在DTB和其他地方作为有希望的治疗药物被探索，这些分析也具有转化潜力。复制机制对扰动的反应与人类健康有关，不同癌细胞类型中特定的细胞周期调节缺陷可能为它们对治疗的敏感性提供线索。我们的研究表明，DNA复制早期阶段的解除管制通过两种不同的途径导致过度复制和随后的基因组不稳定：一种涉及休眠起源的激活，另一种涉及基线起源的过度激活。我们目前正在描述由每种途径激活的复制起源的遗传和表观遗传特性，以及每种途径调节的蛋白质- dna相互作用。使用单纤维分析和基于测序的技术相结合，我们分析了暴露于化疗药物和染色质调节剂后的复制动力学。这些研究有望确定通常参与防止过度复制的染色质靶点，并揭示将代谢状态传递给染色质的信号通路。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，以影响复制景观和细胞对抗癌治疗的反应。我们的研究依赖于我们开发的工具来绘制整个基因组的复制起始位点，并比较具有不同染色质特征的复制起始位点。我们还与NCI内外的其他研究人员合作，以表征癌细胞的遗传和表观遗传特征，并参与将遗传和表观遗传特征与治疗反应联系起来的合作努力。在未来的研究中，我们计划进一步剖析调节染色体复制的分子相互作用。具体来说，使用单纤维分析、生化和计算工具的组合，我们将系统地表征蛋白质-DNA和蛋白质-蛋白质相互作用，这些相互作用介导了RepID、SIRT1和DNA损伤信号通路对染色体复制程序的影响。

项目成果

期刊论文数量（37）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability.

磷酸化的SIRT1与复制起源相关，以防止复制过多并保留基因组稳定性。

DOI：
10.1093/nar/gkx468
发表时间：
2017-07-27
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Utani K;Fu H;Jang SM;Marks AB;Smith OK;Zhang Y;Redon CE;Shimizu N;Aladjem MI
通讯作者：
Aladjem MI

Encounters in Three Dimensions: How Nuclear Topology Shapes Genome Integrity.