Initiation of DNA Replication in Mammalian Cells

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。我们使用两种方法来表征dna -蛋白质在复制起始位点的相互作用。第一种方法利用不同的DNA序列，称为复制因子，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。其中一个复合体包括染色质重塑蛋白，它决定复制时间和转录活性（Mol Cell Biol. 31:3472-84; 2011）。另一个复合体包括RepID，它是ddb1 - cul4相关因子（DCAF）家族的成员，它结合了复制起始位点的子集，并且是在这些位点进行复制所必需的（Nat common . 8;7:11 1748; 2016）。我们的研究表明，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。我们已经描述了RepID与其他蛋白质的相互作用，使用无偏见的方法鉴定了RepID蛋白质伴侣，并确定了RepID中促进dna -蛋白质和蛋白质-蛋白质相互作用的蛋白质结构域。我们的分析表明，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。第二种方法涉及开发工具来绘制整个基因组的复制起始位点，并使用这些工具来分析染色质修饰和转录活性背景下的DNA复制。发展的方法包括大规模并行测序和复制叉进程的单纤维成像。这些程序使我们能够在全基因组水平上研究DNA复制的动力学。使用这种方法，我们可以测试一组复制起始位点是否共享特定的属性——例如，它们是否与特定的染色质特征相关联。我们还可以确定以类似方式响应细胞挑战的起始位点组，并测试是否通过与特定蛋白质（如RepID）的关联来调节不同组的复制起始位点。我们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（Genome Res. 21:1822-32, 2010; Epigenetics and Chromatin . 9:18, 2016）。我们最近对与DNA复制起点相关的染色质修饰的综合分析（表观遗传学和染色质9:18,2016）表明，复制起点的使用随组织类型而变化，与细胞类型特异性复制起点相关的修饰不同。为了促进这些研究，我们开发了一个基于网络的工具（colweb; BMC Genomics 16:142）。2015)，以帮助破译RepID结合位点与表观遗传特征之间的关系。该工具可用于社区支持dna -蛋白质相互作用位点的生物信息学表征。我们还使用全基因组数据来鉴定与复制起始事件相关的DNA和组蛋白修饰。例如，我们观察到复制起始与DNAse超敏感位点和二甲基化组蛋白H3赖氨酸79之间存在很强的关联，这表现出动态的细胞周期分布（PLoS Genet. 9:e1003542, 2013）。我们还证明（核酸研究，出版），磷酸化形式的NAD+依赖的去乙酰化酶，SIRT1，结合潜在的复制起点，并阻止在这些潜在起点的亚群（“休眠起点”）开始复制。在合作研究中，我们描述了晚期复制起源与ORCA/LRWD1之间的相互作用，ORCA/LRWD1是异染色质的一个组成部分（核酸研究45:2490-2502）。在另一项合作研究中，我们使用了复制起始的阶段性等位基因特异性分析来破译复制起始的序列要求（Nature Communications 6:7051）。2015)。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。我们观察到DNA修复内切酶Mus81在没有外源胁迫的情况下调节DNA复制的速度，并且它的存在对于帮助细胞在减缓复制的药物存在下恢复DNA合成是必不可少的（Nature Communications 6:6746）。2015)。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。

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 use two approaches to characterize DNA-protein interactions at replication initiation sites. The first approach utilizes 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 chromatin remodeling proteins that determine both replication timing and transcriptional activity (Mol Cell Biol. 31:3472-84; 2011). Another complex 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 (Nat Commun. 8;7:11748; 2016). Our studies have demonstrated that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. We have characterized RepID interactions with other proteins, identified RepID protein partners using a non-biased approach and pinpointed protein domains within RepID that facilitate DNA-protein and protein-protein interactions. Our analyses demonstrate that 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. The second approach involves developing tools to map replication initiation sites throughout the genome, and using these tools to analyze DNA replication in the context of chromatin modifications and transcriptional activity. The developed methods involve massively parallel sequencing and single-fiber imaging of replication fork progression. These procedures allow us to study the dynamics of DNA replication at the whole-genome level. Using this methodology we can test whether groups of replication initiation sites share specific properties - for example, if they associate with a particular chromatin feature. We can also identify groups of initiation sites that respond in a similar fashion to a cellular challenge, and test whether distinct groups of replication initiation sites are regulated through association with particular proteins (such as RepID). We have generated a comprehensive dataset of replication initiation sites for several human cancer cell lines (Genome Res. 21:1822-32, 2010; Epigenetics and Chromatin 9:18, 2016). Our recent comprehensive analysis of chromatin modifications associated with DNA replication origins (Epigenetics and Chromatin 9:18, 2016) demonstrated that replication origin usage varies with tissue type, with distinct modifications associated with cell-type specific replication origins. To facilitate these studies, we have developed a web-based tool (Coloweb; BMC Genomics 16:142. 2015) to help decipher the relationships among RepID binding sites and epigenetic features. This tool is available to the community to support bioinformatics characterization of DNA-protein interaction loci. We also use genome-wide data to identify DNA and histone modifications that associate with replication initiation events. For example, we observed strong associations between replication initiation and both DNAse hypersensitive sites and dimethylated histone H3 lysine 79, which exhibits a dynamic cell cycle distribution (PLoS Genet. 9:e1003542, 2013). We have also demonstrated (Nucleic Acids Research, in press) that a phosphorylated form of the NAD+-dependent deacetylase, SIRT1, binds potential replication origins and prevents replication from initiating in a subgroup of those potential origins ("dormant origins"). In collaborative studies, we have characterized an interaction between late-replicating origins and ORCA/LRWD1, a component of heterochromatin (Nucleic Acids Research 45:2490-2502). In another collaborative study, we have used phased allele-specific analyses of replication origins to decipher the sequence requirements for replication initiation (Nature Communications 6:7051. 2015). 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 observed that a DNA repair endonuclease, Mus81, modulates the pace of DNA replication in the absence of exogenous stress and that its presence is essential to help cells restore DNA synthesis in the presence of drugs that slow replication (Nature Communications 6:6746. 2015). 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.