The role of nucleases in interstrand crosslink repair

描述(由申请人提供)：对我们细胞遗传物质的破坏可能会产生许多不受欢迎的后果。它可能导致细胞死亡、生长停滞、不适当的生长或突变。这些在机体水平上的结果是基本器官功能衰竭或癌症。罕见的人类遗传病启发了我们，DNA修复的缺乏以及细胞中DNA损伤的持续存在是如何导致这些问题的。我们的实验室研究了两种DNA修复疾病，Fanconi贫血(FA)和核巨大性间质性肾炎(KIN)。FA患者有发育异常，包括骨骼异常和骨髓衰竭，这使得他们无法产生足够的红细胞来输送氧气，无法产生足够的血小板来防止出血，也无法产生足够的白细胞来抵御感染。FA患者ALS有非常高的癌症易感性，包括急性髓细胞性贫血，这种疾病在骨髓衰竭、头颈癌和妇科癌症的环境中矛盾地发生。KIN患者发展为肾衰竭，需要透析和肾移植。虽然罕见，但这些疾病可以作为强大的模型来理解骨髓和肾脏是如何衰竭的，以及当DNA得不到修复时癌症是如何发展的。我们努力了解这些疾病的分子基础、它们之间的联系和区别。尽管这两种疾病的患者表现出不同的健康问题，但患者的细胞缺乏修复一种非常特殊的DNA损伤类型的能力，这种损伤是链间交联，它将两条DNA连接在一起，阻止了它们的分离。这种损害可能是由环境毒素、细胞过程中的代谢物或癌症治疗期间的化疗造成的。在这项授权中，我们建议将注意力集中在参与处理链间交联的核酸酶上。我们已经在国际Fanconi贫血登记中发现了三名Fanconi贫血患者的SLX4突变。与我们的合作者一起，我们描述了KIN患者的FAN1突变。在前两个目标中，我们建议使用患者细胞系来了解这两种疾病的发病机制。利用分子手段，我们想了解SLX4结合的核酸酶以及FAN1的相互作用，它们在细胞周期和不同DNA损伤中的不同要求，以及它们如何与细胞中的其他DNA修复途径进行遗传相互作用。在第三个目标中，我们将采用生化方法来了解这些核酸酶。在进行体外实验时，我们想研究它们是如何处理受损的DNA的。我们的目标是对细胞如何处理交联链有一个详细的了解，希望为治疗应用操纵修复途径。

DESCRIPTION (provided by applicant): Damage to the genetic material of our cells can have many undesired consequences. It may lead to cell death, growth arrest, inappropriate growth or mutations. The outcomes of these on the organismal levels are failure of essential organ function or cancer. Rare human genetic diseases have enlightened us about how lack of DNA repair and thus persistence of DNA damage in our cells leads to these problems. Our laboratory studies two DNA repair diseases, Fanconi anemia (FA) and Karyomegalic Interstitial Nephritis (KIN). Patients with FA have developmental abnormalities including skeletal anomalies and bone marrow failure, which leaves them unable to produce enough red blood cells to carry oxygen, platelets to prevent bleeding or white blood cells to fight off infections. FA patients als have a very high predisposition to developing cancer including acute myelogenous anemia that occurs paradoxically in the setting of the bone marrow failure, head and neck cancers, and gynecologic cancers. KIN patients develop kidney failure and need dialysis and kidney transplantation. Although rare, these diseases can be used as powerful models for understanding how bone marrow and kidneys fail, and how cancer develops when the DNA is not repaired. We strive to understand the molecular underpinnings of these diseases, connections and differences between them. Even though the patients with the two diseases show different health problems, the cells from the patients lack the ability to repair a very particular type of DNA damage, interstrand crosslink, which links the two strands of DNA together precluding their separation. This kind of damage may be caused by environmental toxins, metabolites from cellular processes or by chemotherapy during cancer treatment. In this grant, we propose to concentrate our attention on the nucleases involved in processing of the interstrand crosslinks. We have identified SLX4 mutations in three patients with Fanconi anemia in the International Fanconi anemia registry. With our collaborators, we have described FAN1 mutations in KIN patients. In the first two aims we propose to use the patient cell lines to understand the pathogenesis of the two diseases. Using molecular approaches we want to understand the interaction of SLX4- bound nucleases as well as FAN1, their different requirements across cell cycle and across different DNA lesions and how they genetically interact with other DNA repair pathways in the cell. In the third aim, we will take a biochemical approach to understand these nucleases. Performing in vitro experiments, we want to study how they work on damaged DNA. Our goal is to have a detailed picture of how the cell deals with crosslinks in hopes of manipulating the repair pathways for therapeutic applications.