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，其中一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，我们将描述复制起始位点的蛋白质-DNA相互作用，并确定在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。我们最近的研究已经确定了两个离散的dna -蛋白质复合物在一个复制因子元件。其中一种复合体包括染色质重塑蛋白，它决定复制时间和转录活性。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。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。我们最近的研究为几种人类癌细胞系生成了一个全面的复制起始位点数据集。我们还发现了复制起始与CpG甲基化之间的正相关，复制与高水平转录之间的负相关。DNA复制的时间和地点不仅取决于局部DNA-蛋白质的相互作用，还涉及染色质构象和与远端序列的相互作用。我们之前已经发现了调控远处DNA复制起始的DNA元件，并且还表明复制元件可以影响附近位置的染色质凝聚和基因表达。在当前的回顾期间，我们研究了复制因子元件与人类β -球蛋白（HBB）位点内远端调控序列之间的染色质环相互作用。我们最近发现了一种影响组蛋白H3乙酰化并防止染色质凝聚和基因沉默的复制子相互作用蛋白复合物。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。在接下来的回顾中，我们将继续描述染色质环和组蛋白修饰如何影响复制起始事件的位置和时间。我们还将深入研究复制因子结合蛋白（RepID）。我们将识别涉及RepID的分子相互作用（蛋白质- dna和蛋白质-蛋白质），并表征调节其活性的信号通路。我们将使用这些途径来分离其他蛋白质底物，可能是那些与不同复制因子结合的蛋白质底物。此外，我们将确定涉及RepID（以及结合其他复制子亚群的RepID样蛋白，如果发现）的相互作用如何随着细胞在细胞周期中的进展而被修改。我们在局部和全基因组水平上的联合研究将确定修饰染色质或调节远端相互作用以确定复制起始位点和指示复制时间的蛋白质。我们之前观察到，轻度暴露于复制抑制剂通过涉及癌症易感蛋白BLM解旋酶、Mus81核酸酶和ATR激酶的机制减慢复制。我们最近观察到Mus81内切酶活性也影响DNA复制的正常速度和在无挑战生长期间开始复制的频率。相比之下，ELG1/ATAD5也参与细胞对受干扰复制的反应，不影响复制起始率。这些结果表明，以前被认为是DNA修复专家的酶可能参与了在不受干扰的生长过程中调节DNA复制的监视机制。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。

Within eukaryotic cells, genome replication 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. Some of 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 will 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. Our recent studies 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. 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. 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). Our recent studies generated a comprehensive dataset of replication initiation sites for several human cancer cell lines. We also identified a positive correlation between replication initiation and CpG methylation, and a negative correlation between replication and high levels of transcription. The decision when and where DNA would replicate depends not only on local DNA-protein interactions, but also involves chromatin conformation and interactions with distal sequences. We had previously identified DNA elements that regulate the initiation of DNA replication at a distance, and had also shown that replicator elements can affect chromatin condensation and gene expression at nearby locations. In the current review period we studied chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. We have recently identified a replicator-interacting protein complex that affects histone H3 acetylation and prevents chromatin condensation and gene silencing. We also used 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. For the next review period we will continue to characterize how chromatin loops and histone modifications affect the location and timing of replication initiation events. We will also thoroughly study the replicator-binding protein, RepID. We will identify molecular interactions (protein-DNA and protein-protein) involving RepID and characterize signaling pathways that modulate its activity. We will use these pathways to isolate other protein substrates, possibly those that bind to different sets of replicators. Further, we will determine how interactions involving RepID (and RepID-like protein that bind other subsets of replicators, if discovered) are modified as cells progress through the cell cycle. Our combined studies at the local and whole genome level will identify proteins that modify chromatin or modulate distal interactions to determine replication initiation sites and dictate replication timing. We previously observed that mild exposure to replication inhibitors decelerate replication via mechanisms that involve the cancer-predisposing proteins BLM helicase, Mus81 nuclease, and ATR kinase. We have recently observed that Mus81 endonuclease activity also affects the normal pace of DNA replication and the frequency of replication initiation during unchallenged growth. In contrast, ELG1/ATAD5, which is also involved in cellular responses to perturbed replication, did not affect replication initiation rates. These results imply that enzymes previously thought to be DNA repair specialists may participate in surveillance mechanisms that regulate DNA replication during unperturbed growth. 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. .