Cellular Responses to DNA Damage

我们正在努力建立一个体外系统，用于研究各种诱导人类细胞DNA损伤的检查点激活。其中一些DNA损伤剂被广泛用于化疗。因此，我们的研究重点是这些化疗药物赋予细胞毒性的分子机制，以及癌细胞获得耐受性的途径。作为一个模型系统，我们正在研究由DNA错配修复蛋白MutSalpha和MutLalpha介导的检查点激活对几种DNA损伤剂（包括SN1烷基化剂、氟尿嘧啶和顺铂）的反应。细胞对DNA损伤的反应涉及信号激酶如ATR和ATM的激活。这进而激活包括Chk1和Chk2在内的一系列信号激酶，最终导致细胞周期阻滞在S期和G2/M边界。我们正在纯化参与这种损伤反应的重组人蛋白，目的是确定潜在的分子机制。

We are working to establish an in vitro system for the study of checkpoint activation by various agents that induce DNA damage in human cells. Some of these DNA damaging agents are widely used in chemotherapy. Thus, our research focuses on molecular mechanisms by which these chemotherapeutic agents confer cytotoxicity as well as pathways by which cancer cells achieve tolerance. As a model system, we are investigating checkpoint activation mediated by the DNA mismatch repair proteins MutSalpha and MutLalpha in response to several DNA damaging agents including SN1 alkylators, fluorouracil, and cisplatin. The cellular response to DNA damage involves the activation of signaling kinases such as ATR and ATM. This, in turn, activates a cascade of signaling kinases including Chk1 and Chk2 that ultimately result in cell cycle arrest in S phase and at the G2/M boundary. We are purifying recombinant human proteins involved in this damage response with the goal of defining the underlying molecular mechanism. We are identifying components of key protein complexes that function to license cell cycle arrest in response to DNA damaging agents. This work involves a collaboration with Dr. Dorothy Erie, UNC-Chapel Hill, to use single molecule imaging techniques to characterize protein complexes. We are also examining the role of nuclear membrane proteins in coordinating dynamic changes in nuclear architecture during the cell cycle and in response to DNA damage. We are identifying sites of cell cycle-dependent modification of components of the nuclear pore complex by mass spectroscopy in collaboration with the NIDDK Mass Spectroscopy Core facility. We are using a variety of functional assays to define the roles that these modifications play in response to signals for cell division and DNA damage.