Eukaryotic DNA Alkylation Repair

描述(由申请人提供)：这是一份重新提交的竞争性续签申请，该申请是为了继续一个已有20多年历史的研究真核细胞DNA烷基化修复的项目。我们建议探索一种特定的DNA修复途径，即由烷基腺嘌呤DNA糖基酶修复酶(AAG)启动的碱基切除修复(BER)，在小鼠中是AAG，在人类中是AAG；MPG/MPG和ApNG/APNG)，有助于不同类型的细胞对DNA损伤剂的反应。烷基化试剂具有细胞毒性、致突变性、致裂性、致畸性和致癌性，是环境中最有效和最丰富的一类化学DNA损伤剂。此外，烷化剂作为正常的细胞代谢物存在于细胞内，它们是氧化应激的副产品。此外，一些毒性最大的烷化剂通常用于癌症患者的化疗。在过去的资助期间，我们了解到的最重要的事情之一(除了确定一些新的途径涉及保护细胞免受烷基化试剂的影响)是，在某些类型的细胞中，Aag DNA修复酶的表达，即使是在野生型水平，也会产生与直觉相反的结果，使这些细胞对DNA烷基化损伤的细胞毒性效应极其敏感。在下一个资助期，我们将寻求更全面地了解这些特定细胞类型的哪些特征使得AAG启动的DNA修复对细胞生存如此不利。这将通过追求以下四个具体目标来实现：目标1、2和3将利用干细胞/再生医学领域的最新进展，在体外将小鼠胚胎干细胞(ES)分化为特定分化的造血髓系细胞、视网膜光感受器细胞和小脑颗粒细胞，所有这些细胞在体内都表现出AAG依赖的烷基化敏感性，不像它们的ES细胞前体表现出AAG依赖的烷基化抗性。此外，作为AIMS 1和3的一部分，我们将直接从小鼠身上分离分化和分化的细胞。将测试所有类型的细胞对两种不同的烷化剂和一种非烷化剂紫外线的敏感性。通过分离和表征 目的4)这些表型已从AAG依赖的烷基化抵抗细胞(ES细胞)转变为AAG依赖的烷基化敏感细胞(髓系细胞、光感受器细胞和颗粒神经元细胞)的同基因细胞，我们希望了解如何更好地预测细胞和人对广泛存在于环境中并在临床上常用的烷基化试剂的反应。拟议项目与健康相关的是，所获得的信息将有助于推进我们预防癌症和其他DNA损伤相关疾病的目标，同时也有助于改善这些疾病，特别是癌症在临床上的治疗方式。

DESCRIPTION (provided by applicant): This is a re-submission of a competitive renewal application for the continuation of a more than twenty year old project to study DNA alkylation repair in eukaryotic cells. We propose to explore how one particular DNA repair pathway, namely base excision repair (BER) initiated by the alkyladenine DNA glycosylase repair enzyme (Aag in mice, AAG in humans; a.k.a. Mpg/MPG and Apng/APNG), contributes to the response of different cell types upon exposure to DNA damaging agents. Alkylating agents are cytotoxic, mutagenic, clastogenic, teratogenic and carcinogenic, and they represent one of the most potent and abundant classes of chemical DNA damaging agents in our environment. In addition, alkylating agents are present inside cells as normal cellular metabolites, and they are produced as byproducts of oxidative stress. Moreover, some of the most toxic alkylating agents are commonly used for the chemotherapeutic treatment of cancer patients. In the past funding period one of the most important things we learned (in addition to identifying a number of new pathways involved in protecting cells against alkylating agents) is that in certain cell types, expression of the Aag DNA repair enzyme, even at wild type levels, has the counter-intuitive consequence of rendering those cells extremely sensitive to the cytotoxic effects of DNA alkylation damage. In the next funding period we will seek to understand more fully what features of these specific cell types render Aag-initiated DNA repair so detrimental to cell survival. This will be achieved by pursuing the following four Specific Aims: Aims 1, 2 and 3 will exploit recent advances in the stem cell/regenerative medicine field for the in vitro differentiatin of mouse embryonic stem (ES) cells into specifically differentiated hematopoietic myeloid cells, retinal photoreceptor cells, and cerebellar granule cells, all of which display Aag-dependent alkylation sensitivity in vivo, unlike their ES cell progenitor that displays Aag-dependent alkylation resistance. In addition, as part of Aims 1 and 3, we will directly isolate differentiatig and differentiated cells from mice. All cell types will be tested for their sensitivity to two diffrent alkylating agents, and one non-alkylating agent, ultraviolet light. By isolating and characterizing (Aim 4) these isogenic cells whose phenotype has transitioned from Aag-dependent alkylation resistance (ES cells) to Aag-dependent alkylation sensitivity (myeloid, photoreceptor and granule neuron cells) we hope to learn how to better predict how cells and people will respond to alkylating agents ubiquitously present in the environment, and commonly used in the clinic. The health relatedness of the proposed project is that the information gained will contribute to furthering our goals to prevent cancer and other DNA damage-related diseases, while also contributing to improving how those diseases, especially cancer, are treated in the clinic.