Analysis of Xenopus Np95 during the cell cycle and in DNA damage responses

DNA不断地受到各种不同因素的损伤。如果这种损伤得不到修复，那么突变就会出现。如果任其持续下去，这些突变可能会导致基因组完整性的丧失。为了保护基因组，细胞进化了防止DNA损伤或DNA复制过程中遇到的问题产生突变的机制。这些机制统称为DNA损伤反应，可以检测受损的DNA或停滞的DNA复制，并可以启动一系列反应，如DNA修复、细胞周期停滞(允许修复时间)，或者如果损伤太大，则会导致细胞程序性死亡(细胞凋亡)。DNA损伤反应通路的失败被认为与癌症的发生以及与癌症易感性相关的几种人类遗传病以及发育和神经异常有关。因此，如果要了解癌症等人类疾病和许多人类发育异常，了解这些途径如何运作并影响其他细胞过程是至关重要的。使用在试管中重建细胞周期事件的两栖虫卵的无细胞提取物，我们将研究一种名为Np95的核蛋白的作用。Np95在许多类型的癌细胞中上调，缺乏Np95蛋白的细胞对DNA损伤剂和抑制DNA复制的药物高度敏感。这些观察表明，Np95对于有效的DNA损伤反应也是必要的。我们将使用非洲爪哇无细胞提取系统来确定细胞周期中Np95功能是如何以及何时需要的，以及从鸡蛋提取液中去除Np95是否会影响细胞对DNA损伤的反应。

DNA is constantly subjected to damage caused by a wide variety of different factors. If this damage is left unrepaired then mutations can arise. If allowed to persist these mutations can lead to a loss of genome integrity. In order to protect the genome, cells have evolved mechanisms to prevent DNA damage or problems encountered during DNA replication from generating mutations. These mechanisms collectively known as DNA damage responses, detect damaged DNA or stalled DNA replication and can initiate a series of responses such as DNA repair, cell cycle arrest (allows time for repair), or if damage is too extensive, programmed cell death (apoptosis). Failure of DNA damage response pathways have been implicated in the development of cancer and in several human genetic diseases associated with a predisposition to cancer as well as developmental and neurological abnormalities. Understanding how these pathways operate and impinge on other cellular processes is therefore vital if human diseases such as cancer and many human developmental abnormalities are to be understood. Using cell-free extracts of amphibian eggs that recreate cell cycle events in the test tube we will study the role of a nuclear protein known as Np95. Np95 is upregulated in many types of cancer cell and cells lacking Np95 protein are highly sensitive to DNA damaging agents and drugs that inhibit DNA replication. These observations suggests that Np95 is also necessary for efficient DNA damage responses. We will use the Xenopus cell-free extract system to determine how and when Np95 function is required during the cell cycle and whether removing Np95 from egg extracts affects the cellular response to DNA damage.