S-phase checkpoint and rereplication in mammalian cells

描述（由申请人提供）：真核细胞中的DNA复制受到严格控制，因此每个细胞周期基因组复制一次且仅一次。这种机制对于遗传信息从一代忠实地传递到下一代是极其重要的。染色体DNA的过度复制将导致遗传不稳定，这通常与癌症等人类疾病有关。我们的长期目标是了解如何在哺乳动物细胞中阻止DNA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复制起始的一个关键是通过许可控制机制在复制起始处严格调节复制前复合体（pre- rcs）的组装。我们发现，当许可控制被破坏时，atr介导的检查点被激活，并在抑制DNA复制中发挥关键作用。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区域和修复与复制相关的DNA损伤。我们认为，当许可控制受损时，s期检查点和DNA修复功能对维持基因组稳定性至关重要。在本研究中，我们将研究s期检查点控制和DNA修复活性在抑制DNA复制和修复复制相关DNA损伤中的机制。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期检查点与复制许可控制之间通信的生物学重要性。其次，我们将研究DNA修复机制在去除复制DNA和修复DNA复制过程中出现的DNA双链断裂（DSBs）中的作用。我们还将定义DNA还原过程中用于修复dsb的修复途径。第三，我们将探讨Mre11/Rad50/Nbs1复合体在抑制DNA复制中的作用，并研究其作用机制。DNA重组将不可避免地导致基因组不稳定，这是恶性表型的一个组成部分。最近的观察发现，在肿瘤发生的初始阶段，DNA复制是被诱导的，这突出了复制控制在预防癌症中的重要性。了解reereplication如何在哺乳动物细胞中被阻止，将有助于阐明控制基因组稳定性的细胞机制，并提高我们对癌症病因的理解。

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.