Hydrolytic and Free Radical Mediated DNA Damage

描述(申请人提供)：在过去的一个世纪里，工业化国家的预期寿命大幅增加。预期寿命延长的一个后果是，通常与衰老有关的疾病，包括癌症和神经退行性变，将影响更多的人。癌症的遗传基础可归因于一系列DNA损伤事件，包括点突变、染色体重排和丢失，以及表观遗传变化，如DNA胞嘧啶甲基化模式改变。众所周知，DNA会受到外源化学物质和辐射的破坏。然而，即使在没有外源物质的情况下，细胞DNA也会被持续的氧化、水解和甲基化。据估计，每天形成的内源性DNA损伤的数量约为每个细胞数万个。作为对DNA损伤的反应，细胞有一个复杂的修复路径网络，识别、切除和重建受损部位。众所周知，DNA修复途径的缺陷会显著增加癌症的发病率。人类寿命的延长，以及包括癌症在内的与年龄相关的疾病的增加，突显了了解DNA如何受损以及修复途径如何发挥作用的必要性。我们在这个项目上的努力重点是辐射、致癌物暴露和内源性DNA损伤可能导致的五种嘧啶氧化产物。这些修饰的嘧啶类化合物包括5-羟甲基尿嘧啶(HMU)、5-甲基尿嘧啶(FOU)、5-羟甲基胞嘧啶(HMC)、5-羟基尿嘧啶(HOU)和5-羟基胞嘧啶(HOC)。在之前的资助期间，我们发现了这些修饰的嘧啶类化合物的几个重要的化学性质，我们发现了几个新的和重要的细胞修复酶识别受损碱基的性质。在当前的提案中，我们描述了一系列实验来跟进这些发现。目前建议的具体目标是：1)确定负责修复HMU的蛋白质，并进一步了解细胞对外源HMU修复的反应；2)研究5-甲基胞嘧啶(5mC)氧化的生化后果，包括HMC对DNA-蛋白质相互作用的影响，并进一步研究可能通过其改变甲基化模式的潜在的新的生化途径；3)确定DNA修复糖基酶识别受损的嘧啶的一般机制；以及4)开发一种通用的策略来鉴定和表征尚未确定的DNA修复蛋白。拟议的研究结果将为我们理解DNA损伤及其修复提供重要的新线索。有了这些新的见解，我们可能会学习如何减少导致包括癌症在内的人类疾病的DNA损伤，以及如何利用DNA修复途径中的缺陷或改变来开发更具选择性的化疗。

DESCRIPTION (provided by applicant): Life expectancy in the industrialized world has increased dramatically over the past century. A consequence of the increasing life expectancy is that diseases generally associated with aging, including cancer and neurodegeneration, will affect more individuals. The genetic basis of cancer is attributed to a series of DNA damage events that can include point mutations, chromosome rearrangements and loss, and epigenetic changes such as altered DNA cytosine methylation patterns. DNA is known to be damaged by exogenous chemicals and radiation. However, cellular DNA is continuously oxidized, hydrolyzed and methylated even in the absence of exogenous agents. It is estimated that the number of endogenous DNA lesions formed is on the order of tens of thousands per cell per day. In response to DNA damage, cells have a complex network of repair pathways that recognize, excise and rebuild the damaged sites. Defects in DNA repair pathways are known to increase substantially the incidence of cancer. An increase in human life span over which DNA can be damaged, coupled with an increase in age-associated diseases including cancer, highlight the need to understand how DNA can be damaged and how repair pathways function.The focus of our efforts on this project has been five pyrimidine oxidation products that can result from radiation, carcinogen exposure and endogenous DNA damage. These modified pyrimidines include 5-hydroxymethyluracil (HmU), 5-formyluracil (FoU), 5-hydroxymethylcytosine (HmC), 5-hydroxyuracil (HoU) and 5-hydroxycytosine (HoC). In the previous period of funding, we discovered several important chemical properties of these modified pyrimidines, and we have discovered several new and significant properties of damaged base recognition by cellular repair enzymes.In the current proposal, we describe a series of experiments to follow up on these findings. The specific aims of the current proposal are 1) to identify the proteins responsible for the repair of HmU and to further understand cellular responses to the repair of exogenous HmU, 2) to investigate biochemical consequences of the oxidation of 5- methylcytosine (5mC) including the impact of HmC on DNA-protein interactions and to further investigate a potential novel biochemical pathway through which methylation patterns might be altered, 3) to identify the general mechanisms by which damaged pyrimidines are recognized by DNA repair glycosylases, and 4) to develop a general strategy for the identification and characterization of as yet unidentified DNA repair proteins. The results of the proposed studies should shed significant new light on our understanding of DNA damage and its repair. With these added insights, we may learn how to reduce DNA damage that leads to human diseases including cancer, as well as understand how to exploit defects or alterations in DNA repair pathways for the development of more selective chemotherapy.