S-phase checkpoint and rereplication in mammalian cells

描述(申请人提供)：真核细胞中的DNA复制受到严格控制，因此基因组只复制一次，并且每个细胞周期只复制一次。这种机制对于将遗传信息忠实地从一代传到下一代极其重要。染色体DNA的过度复制将导致遗传不稳定，这通常与人类疾病有关，如癌症。我们的长期目标是了解如何在哺乳动物细胞中防止DNA再复制，以及DNA复制控制的丧失如何导致基因组不稳定和肿瘤发生。控制DNA复制起始的关键之一是通过许可控制机制在复制起始处严格控制复制前复合体(Pre-RCs)的组装。我们发现，当许可控制受到损害时，ATR介导的检查点被激活，并在抑制DNA再复制方面发挥关键作用。我们最近的研究进一步表明，DNA修复机制积极参与去除过度复制的DNA区域和修复与再复制相关的DNA损伤。我们认为，当许可控制受损时，S阶段的检查点和DNA修复功能对于维持基因组的稳定性是至关重要的。在这个提案中，我们将研究S阶段检查点控制和DNA修复活动在抑制DNA再复制和修复与再复制相关的DNA损伤中的作用机制。首先，我们将通过检查点蛋白与许可因子CDT1的直接相互作用，研究S阶段检查点与复制许可控制之间的通信的生物学重要性。其次，我们将研究DNA修复机制在去除DNA再复制和修复DNA双链断裂(DSB)中的作用。我们还将定义在DNA重新复制过程中用于修复DSB的修复途径。第三，我们将探索Mre11/Rad50/Nbs1复合体在抑制DNA再复制中的作用，并探讨其潜在的作用机制。DNA再复制不可避免地会导致基因组的不稳定，这是恶性表型的一个组成部分。最近的观察表明，在肿瘤形成的初始阶段会诱导再复制或非计划的DNA复制，这突显了复制控制在预防癌症中的重要性。了解哺乳动物细胞如何防止再复制将有助于揭示支配基因组稳定性的细胞机制，并提高我们对癌症病因学的理解。 公共卫生相关性：染色体DNA的DNA重复复制或过度复制经常导致基因组不稳定，这与癌症的发展高度相关。因此，了解哺乳动物细胞中DNA再复制是如何被抑制的，对于阐明预防肿瘤发生的机制具有重要意义。拟议的研究将阐明癌症病因学，并最终将有助于开发与基因组不稳定和癌症相关的人类疾病的新治疗干预措施。

DESCRIPTION (provided by applicant): DNA replication in eukaryotic cells is tightly controlled so that the genome is replicated once and only once per cell cycle. Such a mechanism is extremely important for faithful transmission of genetic information from one generation to the next. Over-replication of chromosomal DNA will result in genetic instability, which is often associated with human diseases, such as cancer. Our long-term objectives are to understand how DNA rereplication is prevented in mammalian cells and how loss of DNA replication control would lead to genome instability and tumorigenesis. One key to the control of DNA replication initiation is the tightly regulated assembly of pre- replication complexes (pre-RCs) at replication origins by the licensing control mechanism. We showed that when the licensing control is compromised, the ATR-mediated checkpoint is activated and plays a critical role in the suppression of DNA rereplication. Our recent studies further demonstrate that DNA repair machineries are actively involved in removing over-replicated DNA regions and repairing rereplication-associated DNA lesions. We propose that both S-phase checkpoint and DNA repair functions are critical for the maintenance of genome stability when the licensing control is impaired. In this proposal, we will investigate the mechanisms underlying the S-phase checkpoint control and DNA repair activities in the suppression of DNA rereplication and in the repair of rereplication-associated DNA lesions. First, we will study the biological importance of the communication between the S-phase checkpoint and the replication licensing control through a direct interaction of checkpoint proteins with the licensing factor Cdt1. Second, we will investigate the role of DNA repair mechanisms in the removal of rereplicated DNA and in the repair of DNA double-strand breaks (DSBs) that arise during DNA rereplication. We will also define the repair pathways which are used to repair DSBs during DNA rereplication. Third, we will probe the role of the Mre11/Rad50/Nbs1 complex in the suppression of DNA rereplication and investigate the mechanisms underlying this function. DNA rereplication would inevitably lead to genome instability, which is an integral aspect of the malignant phenotype. Recent observations that rereplication or unscheduled DNA replication is induced at the initial stages of tumorigenesis highlight the importance of replication control in the prevention of cancer. Understanding how rereplication is prevented in mammalian cells will shed light on the cellular mechanisms which govern genome stability and improve our understanding of cancer etiology. PUBLIC HEALTH RELEVANCE: DNA rereplication or over-replication of chromosomal DNA often contributes to genome instability, which is highly associated with cancer development. Therefore, understanding how DNA rereplication is suppressed in mammalian cells is of great importance for clarifying the mechanisms underlying the prevention of tumorigenesis. The proposed studies will shed light on cancer etiology and will ultimately help develop new therapeutic interventions for human diseases associated with genome instability and cancer.