DNA Replication Initiation Sites in Mammalian Cells

项目总结/摘要 重复序列会产生难以复制（DTR）的区域，这些区域会阻止复制，威胁到基因组 完整核糖体DNA（rDNA）、着丝粒α（α）卫星和端粒是重复的DTR区域 其序列形成阻碍复制型DNA聚合酶（Pos）的结构。这个项目的目标是 为了深入了解影响这些不同生物学类别的准确复制的因素， 重要的重复元素。我们建议研究可能影响其复制和关键的因素， 这些机制使这些染色体元件得以稳定维持。在目标1中，我们将研究 跨损伤合成（TLS）Pos在复制rDNA、α-卫星和端粒DTR区域中的作用。证据 在重复DTR基因座的维持中暗示TLS Pos强烈表明TLS Pos在以下方面发挥作用： 使得重复DTR序列能够正常复制，这可能是通过复制性Pol-TLS Pol交换实现的。 我们将确定复制过程中在单个分子上的复制性Pol - TLS Pol交换的程度。 水平在个别活细胞使用先进的新型超分辨率显微镜的方法，我们有 开发我们将确定rDNA、α-卫星和端粒位点的聚合酶交换动力学， 建立交换的要求，包括PCNA的参与及其单泛素化。我们 假设TLS Pos参与rDNA、α-卫星和端粒区域的正常复制， 这些聚合酶因此维持这些基因座的稳定性。我们将通过以下方式来检验这一预测： 确定降低的TLS Pol活性是否损害这些DTR区域在未应激细胞中的稳定性。在 目的2：阐明人类rDNA和α-卫星重复序列的复制程序， 确定影响其复制的功能。我们将采用自然发生的重复位点和异位 引入的染色体携带确定的和独特的可区分的人类rDNA或着丝粒α-卫星 重复作为模型基因座，以建立特定的复制程序。使用这些模型位点，我们将确定 rDNA序列/重复序列排列对它们复制程序的贡献，并分析重复序列的稳定性， 这些基因座来确定特定的序列/排列是否更倾向于不稳定。我们还将 研究微管结合蛋白tau对rDNA复制的潜在影响，因为它与 rDNA已被证明参与复制介导的rDNA不稳定性。Tau还结合着丝粒α- 卫星DNA，可能影响其复制和稳定性。因此，我们将确定tau的影响 rDNA和α-卫星复制程序的表达。我们预计，这些拟议的研究将 大大增加了我们对rDNA、α-卫星和端粒复制的理解，并使我们能够建立新的 复制的范例。此外，随着TLS Pos目前被评估为可药物化的目标， 特别是在癌症治疗中，我们的研究将为TLS的意外影响提供重要的见解 Pol靶向rDNA、α-卫星和端粒复制和稳定性。

PROJECT SUMMARY / ABSTRACT Repetitive sequences create difficult-to-replicate (DTR) regions that can stall replication, threatening genomic integrity. Ribosomal DNA (rDNA), centromeric alpha (α) satellites and telomeres are repetitive DTR regions whose sequences form structures that impede replicative DNA Polymerases (Pols). The goal of this project is to gain insights into the factors that affect the accurate replication of these distinct classes of biologically important repetitive elements. We propose to examine factors that can impact their replication and key mechanisms that allow these chromosomal elements to be stably maintained. In Aim 1 we will investigate the role of translesion synthesis (TLS) Pols in replicating rDNA, α-satellite and telomere DTR regions. Evidence implicating TLS Pols in the maintenance of repetitive DTR loci strongly suggests TLS Pols play a role in enabling normal replication of repetitive DTR sequences, likely through replicative Pol – TLS Pol exchange. We will determine the extent of replicative Pol – TLS Pol exchange during replication at the single molecule level in individual live cells using a cutting-edge novel super resolution microscopy approach we have developed. We will determine the dynamics of polymerase exchange at rDNA, α-satellite and telomere loci and establish requirements for exchange including the involvement of PCNA and its monoubiquitination. Our hypothesis that TLS Pols participate in the normal replication of rDNA, α-satellite and telomere regions predicts that these polymerases consequently maintain the stability of these loci. We will test this prediction by determining if reduced TLS Pol activity compromises the stability of these DTR regions in unstressed cells. In Aim 2, we will elucidate the replication programs of the human rDNA and α-satellite repeated sequences and determine features that impact their replication. We will employ naturally occurring repeat loci and ectopically introduced chromosomes carrying defined and uniquely distinguishable human rDNA or centromere α-satellite repeats as model loci to establish specific replication programs. Using these model loci, we will determine the contribution of rDNA sequence/repeat arrangement on their replication program and analyze repeat stability in these loci to establish whether specific sequences/arrangements are more prone to instability. We will also investigate the potential impact of the microtubule binding protein tau on rDNA replication, as its binding to rDNA has been shown to be involved in replication-mediated rDNA instability. Tau also binds to centromeric α- satellite DNA, potentially affecting its replication and stability. Thus, we will determine the effect of tau expression on rDNA and α-satellite replication programs. We expect that these proposed studies will both greatly increase our understanding of rDNA, α-satellite and telomere replication and allow us to establish new paradigms for their replication. Furthermore, with TLS Pols currently being evaluated as druggable targets, particularly in cancer treatment, our studies will provide essential insight on the unanticipated impact of TLS Pol targeting on rDNA, α-satellite and telomere replication and stability.