DNA Damage Checkpoints

描述：(申请人提供)人53BP1(P53肿瘤抑制因子结合 蛋白1)具有与酿酒酵母Rad9高度同源的BRCT结构域 (Sc_Rad9)和S.pombe Rhp9/Crb2(Sp_Rhp9/Crb2)DNA损伤检查点蛋白。 为了研究53BP1是否在细胞对DNA损伤的反应中发挥作用，我们 用免疫荧光法检测其在细胞内的定位。未经治疗的 原代细胞和U2OS骨肉瘤细胞，53BP1呈弥漫性核 染色；但在暴露后迅速定位于离散的核病灶 细胞对电离辐射(IR)的敏感性。有几条证据表明，这些 病灶是DNA双链断裂(DSB)的部位。基于这些初步的 结果及53BP1与酵母DNA损伤的序列同源性 检查点蛋白SC_Rad9和Sp_Rhp9/Crb2，我们假设53BP1是一个 早期参与细胞对DNA双链断裂的反应，并在 DNA损伤检查点响应。为了检验这些假设，我们提出了 以下具体目标：一.阐明53BP1的靶向机制 至JR诱导的病灶。我们将目标为53BP1的结构域映射到JR-Induced Foci并探索该结构域针对这些Focus的机制， 包括检查该结构域是否与DNA或染色质结合。2.确定 53BP1是否发挥DNA损伤检查点的作用。我们将研究DNA 损伤检查点反应(细胞周期停滞、细胞凋亡、放射敏感性、 ATM、ATR、Chk1、Chk2、P53和CDC25C)在53BP1细胞中的激活 功能被上调或下调。我们还将研究物理 肺癌组织中53BP1和P53的相互作用及其对P53激活的意义 对DNA损伤的反应。 3.与正常细胞不同，正常细胞只有在对IR做出反应时才有53BP1焦点，许多 即使在没有外源DNA的情况下，人类癌细胞也显示出53BP1病灶 破坏性因素。我们将对非放射癌症中的53BP1病灶进行表征 细胞来确定它们是否代表DNA双链断裂的位置。我们还将检查 我们是否可以利用53BP1正常和正常行为之间的差异 肿瘤细胞特异靶向肿瘤细胞。 总体而言，这项建议旨在帮助我们理解DNA损伤 检查点，并描述一种新的、潜在的重大差异 在正常细胞和癌细胞之间。

DESCRIPTION: (provided by applicant) Human 53BP1 (p53 tumor suppresor binding protein 1) has BRCT domains with high homology to the S. cerevisiae Rad9 (Sc_Rad9) and S. pombe Rhp9/Crb2 (Sp_Rhp9/Crb2) DNA damage checkpoint proteins. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; but localized to discreet nuclear foci rapidly after exposure of the cells to ionizing radiation (IR). Several lines of evidence suggest that these foci are sites of DNA double-strand breaks (DSBs). Based on these preliminary results and the sequence homology between 53BP1 and the yeast DNA damage checkpoint proteins Sc_Rad9 and Sp_Rhp9/Crb2, we hypothesize that 53BP1 is an early participant in the cellular response to DNA DSBs and has a role in the DNA damage checkpoint response. To test these hypotheses, we propose the following Specific Aims: I. Elucidate the mechanism by which 53BP1 is targeted to JR-induced foci. We will map the domain that targets 53BP1 to JR-induced foci and explore the mechanism by which this domain is targeted to these foci, including examining whether this domain binds to DNA or chromatin. 2. Determine whether 53BP1 functions as a DNA damage checkpoint. We will study the DNA damage checkpoint response (cell cycle arrest, apoptosis, radiosensitivity, activation of ATM, ATR, Chk1, Chk2, p53 and Cdc25C) in cells in which 53BP1 function is upregulated or downregulated. We will also study the physical interaction of 53BP1 and p53 and its significance for activation of p53 in response to DNA damage. 3. Unlike normal cells, which have 53BP1 foci only in response to IR, many human cancer cell lines show 53BP1 foci even in the absence of exogenous DNA damaging agents. We will characterize the 53BP1 foci in non-irradiated cancer cells to determine, if they represent sites of DNA DSBs. We will also examine whether we can exploit the difference in 53BP1 behavior between normal and cancer cells to specifically target tumor cells. Overall, this proposal aims to contribute to our understanding of DNA damage checkpoints and to characterize a novel, potentially significant, difference between normal and cancer cells.