Initiation of DNA Replication in Mammalian Cells

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。许多导致异常增殖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。由于来自细胞周期调节网络的信号最终汇聚在染色质上，我们的目标是了解复制事件的位置和时间如何与染色质上的特定修饰相关联，以及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解剖促进复制的DNA序列和结合这些序列的蛋白质，以努力了解细胞如何决定何时何地开始复制。其次，我们使用大规模平行测序和复制成像方法来研究整个基因组中DNA复制的动力学，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，并分析了一个基因组位点，即11号染色体上的人类β -珠蛋白位点上复制子活性的遗传决定因素（Aladjem， Rodewald等人1998；Wang， Lin等人2004；Wang， Lin等人2006）。我们观察到，并非所有潜在的复制因子在每个细胞周期中都启动复制，表观遗传过程在决定是否以及何时在每个s期使用特定的复制因子方面发挥作用(（Fu, Wang et al. 2006)，详见（Aladjem 2007））。最近，我们在复制子序列中发现了蛋白质- dna相互作用的位点，并观察到复制子的活性取决于这些蛋白质结合位点的完整性。今年，我们在复制子RepP中发现了两个离散的dna -蛋白质复合物，RepP位于人类β -珠蛋白位点。一个repp相关复合体包括染色质重塑蛋白，影响邻近序列的复制时间和转录活性，并介导复制子与远端基因座控制区的相互作用（Huang, Fu et al. 2011）。另一个repp相关复合体（未发表）包括DCAF蛋白家族的成员，与复制前复合体相互作用，对DNA复制的起始至关重要。这些发现表明，起始事件的位置取决于细胞周期调节蛋白与潜在复制子中的序列模块的相互作用，并且调节起始的蛋白以合作和组合的方式起作用。这种相互作用可能是哺乳动物染色体起始位点可变使用的基础，并决定了复制的时间。为了评估复制如何与其他基因座的基因表达相关联，我们确定了人类细胞中复制起始事件的全基因组分布（Martin, Ryan et al. 2011）。全基因组研究创建的数据集涵盖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。我们发现，在转录水平中等的基因组区域，复制起始事件的频率增加，但在转录率高的基因中，起始频率降低。与此一致的是，高分辨率图谱显示，复制起始事件被排除在启动子区域之外，并在转录启动子的下游富集。我们还发现起始事件的频率受到染色质缩聚和CpG序列甲基化的影响。这些发现使我们提出了一个模型，表明复制子序列在协调复制、转录和染色质凝聚中的作用。此外，我们利用DNA复制的单纤维分析来确定参与细胞对复制应激反应的新途径。我们发现，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。在该通路的早期阶段，抑制剂诱导短暂的DNA断裂，并在涉及DNA- pk和XRCC4的反应中通过非同源末端连接（NHEJ）途径迅速修复。DNA断裂的快速修复可以防止细胞周期停滞，尽管复制叉进程的速率发生了微小的变化（Shimura, Martin et al. 2007; Shimura, Torres et al. 2008）。在其他研究中，我们与Pommiers博士的LMP小组合作，表征了癌细胞对干扰DNA复制的药物的反应（Seiler, Conti et al. 2007; Conti, Leo et al. 2010）。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。目前的研究主要集中在Mus81内切酶途径对DNA起始频率和复制速度的影响上。Aladjem, m.i.（2007）。“环境中的复制：后生动物DNA复制模式的动态调节。”学报，8(8):588-600。Aladjem， M. I.， L. W. Rodewald等（1998）。“哺乳动物复制因子在人类-珠蛋白位点的基因解剖”科学281(5379):1005-1009。Conti， C.， E. Leo等人（2010）。抑制癌细胞中的组蛋白去乙酰化酶可以减缓复制分叉，激活休眠起源，并诱导DNA损伤。巨蟹座研究70(11):4470-4480。傅海华，王磊，等。（2006）。“用人类复制因子防止基因沉默。”生物工程学报，24(5):572-576。黄磊，傅慧，等。（2011）。由SWI/SNF、MeCP1和hnRNP C1/C2组成的复制子结合复合体预防转录沉默。中国生物医学工程学报，31(6):344 - 344。Martin， M. M. Ryan等（2011）。“高转录区域复制起始事件的全基因组耗竭。”Genome Res. Seiler， J. A.， C. Conti等（2007）。s期内检查点影响DNA复制起始和延伸：单细胞和DNA纤维分析。中国生物医学工程学报，27(6):444 - 444。Shimura， T.， M. M. Martin等（2007）。“DNA- pk参与修复由DNA复制减速引起的DNA断裂的短暂激增。”中国生物医学工程学报，32(3):663 - 668。Shimura， T.， M. J. Torres等（2008）。“布卢姆氏综合症解旋酶和Mus81是在DNA复制压力下诱导瞬时双链DNA断裂所必需的。”中国生物医学工程学报，32(4):444 - 444。王磊，林春明，等（2004）。人类-珠蛋白复制起始区由两个模块独立的复制因子组成。中国生物医学工程学报，24(8):393 - 393。王磊，林春明，等。（2006）。合作序列模块决定了人类-珠蛋白位点的复制起始位点。中国生物医学工程学报，25(6):344 - 344。

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 proliferation 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. Because signals from cell cycle regulatory networks ultimately converge on chromatin, we aim to understand how the location and the timing of replication events are linked to particular modifications on chromatin and how 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 dissect DNA sequences that facilitate replication and proteins that bind such sequences in an effort to understand how cells determine where and when replication initiates. Second, we use massively parallel sequencing and replication imaging approaches to study the dynamics of DNA replication throughout the genome 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 essential for replicator activity in one genomic locus, the human beta globin locus on chromosome 11 (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 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)) . Recently, we identified sites of protein-DNA interactions in replicator sequences and observed that replicator activity depends on the integrity of these protein-binding sites. This year, we identified two discrete DNA-protein complexes within a replicator, RepP, located at the human beta globin locus. One RepP-associated complex includes chromatin-remodeling proteins, affects replication timing and transcriptional activity in adjacent sequences and mediates the interaction of the replicator with a distal locus control region (Huang, Fu et al. 2011). The other RepP-associated complex (unpublished) includes a member of the DCAF protein family, interacts with the pre-replication complex and is essential for initiation of DNA replication. These findings imply that the locations of initiation events depend on interactions of cell cycle regulatory proteins with sequence modules that reside within potential replicators and that the proteins that regulate initiation function in a cooperative and combinatorial manner. Such interactions may underlie the variable use of initiation sites observed in mammalian chromosomes and determine the timing of replication. To assess how replication associates with gene expression in other loci, we determined the genome-wide distribution of replication initiation events in human cells (Martin, Ryan et al. 2011). The dataset created by the genome wide studies encompasses the locations of replication initiation sites throughout the entire non-repetitive genomes of the analyzed transformed and non-transformed cells. We found that the frequency of replication initiation events increased in genomic regions that were transcribed in moderate levels but that initiation frequency was reduced in genes with high transcription rates. In concordance, high-resolution mapping showed that replication initiation events were excluded from promoter regions and enriched immediately downstream of transcribed promoters. We also found that the frequency of initiation events was affected by chromatin condensation and methylation at CpG sequences. These findings lead us to propose a model suggesting a role for replicator sequences in coordinating replication, transcription and chromatin condensation. In addition, we utilized single fiber analyses of DNA replication to identify a new pathway involved in the cellular response to replicative stress. We showed 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. In early stages of the pathway, inhibitors induce transient DNA breaks that are rapidly repaired by the non-homologous end-joining (NHEJ) pathway in a reaction involving DNA-PK and XRCC4. Rapid repair of the DNA breaks prevents cell cycle arrest despite minor changes in the rate of replication fork progression(Shimura, Martin et al. 2007; Shimura, Torres et al. 2008). In other studies, we collaborated with Dr. Pommiers group in LMP to characterize the response of cancer cells to drugs that perturb DNA replication (Seiler, Conti et al. 2007; Conti, Leo et al. 2010). 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. Current studies focus on the impact of the Mus81 endonuclease pathway on the frequency of initiation and the pace of DNA replication. Aladjem, M. I. (2007). "Replication in context: dynamic regulation of DNA replication patterns in metazoans." Nat Rev Genet 8(8): 588-600. Aladjem, M. I., L. W. Rodewald, et al. (1998). "Genetic dissection of a mammalian replicator in the human beta-globin locus." Science 281(5379): 1005-1009. Conti, C., E. Leo, et al. (2010). "Inhibition of histone deacetylase in cancer cells slows down replication forks, activates dormant origins, and induces DNA damage." Cancer Res 70(11): 4470-4480. Fu, H., L. Wang, et al. (2006). "Preventing gene silencing with human replicators." Nat Biotechnol 24(5): 572-576. Huang, L., H. Fu, et al. (2011). "Prevention of Transcriptional Silencing by a Replicator-Binding Complex Consisting of SWI/SNF, MeCP1, and hnRNP C1/C2." Mol Cell Biol 31(16): 3472-3484. Martin, M. M., M. Ryan, et al. (2011). "Genome-wide depletion of replication initiation events in highly transcribed regions." Genome Res. Seiler, J. A., C. Conti, et al. (2007). "The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses." Mol Cell Biol 27(16): 5806-5818. Shimura, T., M. M. Martin, et al. (2007). "DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication." J Mol Biol 367(3): 665-680. Shimura, T., M. J. Torres, et al. (2008). "Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress." J Mol Biol 375(4): 1152-1164. Wang, L., C. M. Lin, et al. (2004). "The human beta-globin replication initiation region consists of two modular independent replicators." Mol Cell Biol 24(8): 3373-3386. Wang, L., C. M. Lin, et al. (2006). "Cooperative sequence modules determine replication initiation sites at the human beta-globin locus." Hum Mol Genet 15(17): 2613-2622.