Initiation of DNA Replication in Mammalian Cells

哺乳动物细胞中 DNA 复制的启动

• 批准号: 8552687
• 负责人: mirit aladjem
• 金额: $ 117.41万
• 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/8552687
• 关键词: Affect; Antineoplastic Agents; Binding; Binding Proteins; Biochemical Genetics; Bloom syndrome protein; Cancer Biology; Cancer cell line; Cell Cycle; Cell division; Cells; Chromatin; Chromosome Condensation; Chromosomes; Complex; Cues; DNA; DNA Repair; DNA Sequence; DNA biosynthesis; DNA-Protein Interaction; Data; Data Set; Distal; Dose; Drug Delivery Systems; Drug resistance; Effectiveness; Environment; Epigenetic Process; Eukaryotic Cell; Event; Exhibits; Exonuclease; Fiber; Future; G1 Phase; Gene Expression; Genetic; Genetic Determinism; Genetic Transcription; Genome; Genomic Instability; Genomics; Goals; Growth; Histone H3; Human; Image; Laboratories; Lead; Lesion; Link; Location; Locus Control Region; Lysine; Malignant Neoplasms; Mammalian Cell; Maps; Metabolic; Methodology; Mining; Modification; Molecular; Mutate; National Heart, Lung, and Blood Institute; Nature; Normal Cell; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Play; Process; Protein Binding; Protein Family; Proteins; Publishing; Recruitment Activity; Regulatory Pathway; Replication Initiation; Replication Origin; Replication-Associated Process; Role; S Phase; Signal Transduction; Site; Staging; Stress; Time; beta Globin; cancer cell; cell growth; cell transformation; chromatin modification; chromatin remodeling; genome wide association study; helicase; inhibitor/antagonist; insight; killings; novel; nuclease; prevent; programs; protein complex; replicator; response; tool

Loss of genetic control causing inappropriate and excessive proliferation is a hallmark of cancer cells. Many of the defective regulatory pathways that lead to aberrant cell growth converge on molecular events that facilitate DNA replication. Replication regulatory pathways can provide good targets for synthetic lethality approaches that specifically kill cancer cells, but replication problems that go undetected can affect genomic integrity, triggering genomic instability that eventually might result in cancer drug resistance. Hence, many anti-cancer drugs target various aspects of DNA replication and the effectiveness of such drugs critically depends on the nature of the lesions affected in particular cancers. The DNA replication group at LMP aims to gain a detailed understanding of the early stages of the replication process in the context of chromatin and cell cycle signaling and investigate how the process is controlled during normal and perturbed growth in cancer and non-cancer cells. Eukaryotic cells start chromosomal replication from multiple regions (replication origins) on each chromosome to duplicate the entire genome prior to each cell division. Replication initiation events occur in precise order during the S-phase of the cell cycle and are subject to strict regulatory processes that are often dysregualted in cancer. Studies in the DNA replication group have previously revealed some of the principles by which the location and the timing of replication initiation events are determined and started to decipher how replication is linked to distinct chromatin modifications. Current studies aim to identify components of the cell cycle signaling network that interact with proteins found on replication origins during normal and perturbed growth and ask how DNA replication coordinates with other chromatin transactions such as transcription, DNA repair and chromosome condensation. To that end, we take two complementary approaches. First, we use biochemical and genetic approaches to identify and characterize DNA sequences that facilitate replication and then use those sequences as baits for proteins that bind such sequences to regulate DNA replication. Second, we use massively parallel sequencing and replication imaging methodology to study the genome-wide dynamics of DNA replication and determine how replication patterns respond to alterations in gene expression, chromatin modifications and drugs that perturb replication. For the first approach, we study DNA sequences (termed replicators) that facilitate initiation of DNA replication at their endogenous chromosomal sites or when they are removed from their endogenous location and transferred to ectopic chromosomal sites. In previous studies, we have identified replicator sequences in mammalian cells and dissected the genetic determinants of replicator activity in two such replicators (Aladjem, Rodewald et al. 1998; Wang, Lin et al. 2004; Wang, Lin et al. 2006). We have observed that not all potential replicators initiate replication during each cell cycle and that epigenetic processes and distal chromatin interactions play a role in determining if and when a particular replicator will be used during each S-phase ((Fu, Wang et al. 2006) reviewed in (Aladjem 2007) and in (Conner and Aladjem 2012)) . Recently, we used the replicator we have identified (RepP) as a bait to identify two discrete DNA-protein complexes. One RepP-associated complex includes chromatin-remodeling proteins and affects replication timing and transcriptional activity (Huang, Fu et al. 2011). The other complex is required for DNA replication (Huang et al., submitted) and includes a novel protein termed Rep-ID. We have characterized Rep-ID interaction with chromatin to reveal that Rep-ID binds chromatin in early G1 phase and dissociates from chromatin as DNA replication proceeds. Importantly, Rep-ID deficient cells exhibit fewer replication initiation sites and aberrant replication, suggesting that Rep-ID is an important determinant of replication initiation events. Next, we will ask how Rep-ID interactions are altered as cells progress through the cell cycle and whether those chromatin interactions would change in normal and cancer cells exposed to environmental challenges and anti-cancer drugs. For the second approach, we have developed tools for mapping replication initiation sites on a genome scale and mining those data and correlate with other chromatin features. Last year we have published the first comprehensive mapping of replication initiation sites in mammalian cells (Martin, Ryan et al. 2011). The dataset created by these studies encompasses the locations of replication initiation sites throughout the entire non-repetitive genomes of the analyzed transformed and non-transformed cells. Combined with the first approach, we have used massively parallel sequencing to analyze the binding patterns of Rep-ID. These studies revealed that Rep-ID preferentially associates with replication origins and the likelihood of binding to Rep-ID decreases with the distance from replication initiation sites and demonstrated that this novel protein binds a subset of replication initiation sites (Huang et al., submitted). We have also collaborated with Keji Zhao (NHLBI) to identify a chromatin modification, trimethylation of histone H3 on lysine 79, that associates with replication initiation sites and prevents over-replication (Fu et al., submitted). Our studies revealed that the decision when and where DNA would replicate on chromatin depends not only on DNA-protein interactions at the local level but also on interactions at a distance. Our recent findings suggest that at the human beta globin locus Rep-ID participates in a complex between the replicator and the locus control region, which is essential for initiation of DNA replication (Huang et al., submitted). This observation proposes a hypothesis for the mechanism underlying the role of distal interactions in regulating DNA replication in metazoan loci. We anticipate that Rep-ID is the first of a family of proteins that recruit the replication machinery to specific replicators and modulate DNA replication in metazoans. Finding replicator-binding proteins that determine whether initiation occurs at particular sites is critical to the understanding of how cell cycle regulatory network interact with chromatin to start replication. To investigate how cells respond to perturbed replication, we utilized single fiber analyses of DNA replication to identify a new pathway involved in the cellular response to replicative stress. We have shown previously that low non-toxic doses of replication inhibitors decelerate replication by a mechanism involving the cancer-predisposing protein BLM helicase, Mus81 nuclease and ATR kinase (Shimura, Martin et al. 2007; Shimura, Torres et al. 2008). Currently we use a mutated Mus81 protein that does not contain an active exonuclease to ask whether the exonuclease activity of Mus81 participates in the regulating the pace of DNA replication and in the response of cells to perturbation of the replication program by anti-cancer drugs. We have expanded the replication initiation site dataset to include several cancer cell lines and cells in which the exonuclease activity of Mus81 was compromised (Fu et al., submitted). The combination of genome-scale sequencing of replication initiation sites and single fiber analyses provide important insights into the organization of replication initiation events and the cellular responses to signals that might perturb DNA replication. Future studies will reveal how the replication landscape would change in normal and cancer cells exposed to environmental challenges and anti-cancer drugs.

失去基因控制导致不适当和过度的增殖是癌细胞的一个标志。许多导致异常细胞生长的有缺陷的调控途径集中在促进DNA复制的分子事件上。复制调控途径可以为专门杀死癌细胞的合成致命方法提供良好的靶点，但未被发现的复制问题可能影响基因组完整性，引发基因组不稳定，最终可能导致癌症耐药性。因此，许多抗癌药物靶向DNA复制的各个方面，这些药物的有效性在很大程度上取决于特定癌症所影响的病变的性质。LMP的DNA复制小组旨在详细了解染色质和细胞周期信号传导背景下复制过程的早期阶段，并研究在癌细胞和非癌细胞的正常和受干扰的生长过程中如何控制这一过程。真核细胞从每条染色体上的多个区域（复制起点）开始染色体复制，在每次细胞分裂之前复制整个基因组。复制起始事件在细胞周期的s期以精确的顺序发生，并受到严格的调控过程，而这些过程在癌症中经常失调。先前对DNA复制组的研究揭示了一些决定复制起始事件的位置和时间的原理，并开始破译复制是如何与不同的染色质修饰联系在一起的。目前的研究旨在确定细胞周期信号网络的组成部分，这些组成部分在正常和受干扰的生长过程中与复制起源上发现的蛋白质相互作用，并询问DNA复制如何与其他染色质交易（如转录、DNA修复和染色体凝聚）协调。为此，我们采取了两种互补的方法。首先，我们使用生化和遗传方法来鉴定和表征促进复制的DNA序列，然后使用这些序列作为结合这些序列的蛋白质的诱饵来调节DNA复制。其次，我们使用大规模平行测序和复制成像方法来研究DNA复制的全基因组动力学，并确定复制模式如何响应基因表达、染色质修饰和干扰复制的药物的改变。对于第一种方法，我们研究了DNA序列（称为复制子），这些DNA序列有助于在其内源性染色体位点开始DNA复制，或者当它们从内源性位置移除并转移到异位染色体位点时。在之前的研究中，我们已经确定了哺乳动物细胞中的复制子序列，并分析了两种复制子中复制子活性的遗传决定因素（Aladjem， Rodewald等人1998年；Wang， Lin等人2004年；Wang， Lin等人2006年）。我们观察到，并非所有潜在的复制子在每个细胞周期中都启动复制，表观遗传过程和远端染色质相互作用在决定特定复制子在每个s期是否以及何时被使用方面发挥着作用(（Fu， Wang等人，2006)，详见（Aladjem 2007）和（Conner and Aladjem 2012））。最近，我们使用我们已经鉴定的复制子（RepP）作为诱饵来鉴定两个离散的dna -蛋白质复合物。一种repp相关复合体包括染色质重塑蛋白，并影响复制时间和转录活性（Huang, Fu et . 2011）。另一个复合体是DNA复制所必需的（Huang et al.，提交），包括一种称为Rep-ID的新蛋白质。我们描述了Rep-ID与染色质的相互作用，揭示了Rep-ID在G1期早期与染色质结合，并随着DNA复制的进行与染色质分离。重要的是，Rep-ID缺陷细胞表现出较少的复制起始位点和异常复制，这表明Rep-ID是复制起始事件的重要决定因素。接下来，我们将询问Rep-ID相互作用如何随着细胞在细胞周期中的进展而改变，以及这些染色质相互作用是否会在暴露于环境挑战和抗癌药物的正常细胞和癌细胞中发生变化。对于第二种方法，我们已经开发了在基因组尺度上绘制复制起始位点的工具，并挖掘这些数据并与其他染色质特征相关联。去年，我们发表了首个哺乳动物细胞复制起始位点的综合图谱（Martin, Ryan et al. 2011）。这些研究创建的数据集包含了被分析转化和非转化细胞的整个非重复基因组的复制起始位点位置。结合第一种方法，我们使用大规模并行测序来分析Rep-ID的结合模式。这些研究表明，Rep-ID优先与复制起始位点相关，与Rep-ID结合的可能性随着距离复制起始位点的距离而降低，并证明这种新蛋白结合了复制起始位点的子集（Huang等人，提交）。我们还与Keji Zhao （NHLBI）合作鉴定了一种染色质修饰，即赖氨酸79上组蛋白H3的三甲基化，该修饰与复制起始位点相关并防止过度复制（Fu等人，已提交）。我们的研究表明，DNA在染色质上复制的时间和位置不仅取决于DNA-蛋白质在局部水平上的相互作用，还取决于远距离的相互作用。我们最近的研究结果表明，在人类β -珠蛋白位点，Rep-ID参与复制子和位点控制区之间的复合物，这对于DNA复制的起始至关重要（Huang et al.，提交）。这一观察结果为后生动物位点中调节DNA复制的远端相互作用的机制提出了一种假设。我们预计Rep-ID是第一个将复制机制招募到特定复制体并调节后生动物DNA复制的蛋白质家族。发现决定起始是否发生在特定位点的复制子结合蛋白对于理解细胞周期调节网络如何与染色质相互作用以开始复制至关重要。为了研究细胞如何对受干扰的复制做出反应，我们利用DNA复制的单纤维分析来确定细胞对复制应激反应的新途径。我们之前已经证明，低剂量的无毒复制抑制剂通过一种涉及癌症易感蛋白BLM解旋酶、Mus81核酸酶和ATR激酶的机制减缓复制（Shimura， Martin等人，2007;Shimura， Torres等人，2008）。目前，我们利用一种不含活性外切酶的突变Mus81蛋白来研究Mus81的外切酶活性是否参与了DNA复制速度的调节以及细胞对抗癌药物干扰复制程序的反应。我们已经扩展了复制起始位点数据集，以包括几种癌细胞系和Mus81外切酶活性受损的细胞（Fu等人，提交）。复制起始位点的基因组尺度测序和单纤维分析的结合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了重要的见解。未来的研究将揭示暴露于环境挑战和抗癌药物的正常细胞和癌细胞的复制景观将如何变化。