Tyrosyl-DNA phosphodiesterase and oxidative DNA damage

描述(申请人提供)：当DNA拓扑异构酶I未能结合形成的瞬时DNA链断裂时，拓扑异构酶将通过酪氨酸键不可逆地连接到3‘DNA末端。然后，这种连接必须被酪氨酸I-DNA磷酸二酯酶(Tdp1)切割，才能修复断裂。人类Tdp1基因缺失与遗传性脊髓小脑性共济失调伴轴索神经病(SCAN1)有关，其临床特征类似于其他与氧化应激相关的共济失调。这些相似性，再加上Tdp1还可以从自由基介导的DNA链断裂的3‘端去除乙醇酸部分，表明Tdp1可能参与了DNA氧化损伤的修复，如果不能修复这种损伤，可能会导致SCAN1的神经元功能障碍。为了验证这些假说，将检测来自SCAN1患者的Tdp1缺陷的淋巴母细胞的细胞毒性以及3‘-磷酸乙醇酸终止的DNA双链断裂的修复。我们将在正常细胞和Tdp1缺陷细胞中比较氧化DNA对端粒DNA的损伤以及端粒的结构和功能。将产生常规的和有条件的Tdp1基因敲除小鼠，并对其进行广泛的鉴定。这将包括对共济失调或其他行为功能障碍的发展以及在存在和不存在氧化应激情况下的神经元凋亡的评估。肿瘤发病率将在未经治疗的动物和暴露于氧化应激的动物中的多个部位确定，Tdp1缺乏对肿瘤进展的影响将在易患癌前前列腺癌的小鼠中进行评估。此外，还将监测老鼠是否有加速衰老的迹象。这些研究旨在阐明Tdp1缺乏导致SCAN1的机制，并可能有助于阐明氧化应激在其他各种神经疾病、癌症和衰老中的作用。

DESCRIPTION (provided by applicant): When transient DNA strand breaks formed by DNA topoisomerase I fail to religate, the topoisomerase becomes irreversibly attached to the 3' DNA end via a tyrosyl linkage. This linkage must then be cleaved by tyrosyI-DNA phosphodiesterase (Tdp1) in order to allow repair of the break. Human deficiency in Tdp1 has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), the clinical features of which are similar to those of other ataxias associated with oxidative stress. These similarities, combined with the finding that Tdp1 also removes glycolate moieties from 3' ends of free radical-mediated DNA strand breaks, suggest that Tdp1 may be involved in repair of oxidative DNA damage, and that failure to repair such damage may lead to neuronal dysfunction in SCAN1. In order to test these hypotheses, cytotoxicity as well as repair of 3'-phosphoglycolate-terminated DNA double-strand breaks will be examined in Tdp1-deficient lymphoblastoid cells, derived from SCAN1 patients. Oxidative DNA damage to telomeric DNA, as well as telomere structure and function, will be compared in normal and Tdp1-deficient cells. Both conventional and conditional Tdp1 knockout mice will be generated and extensively characterized. This will include an assessment for the development of ataxia or other behavioral dysfunctions as well as for neuronal apoptosis both in the presence and absence of oxidative stress. Tumor incidence will be determined at a number of sites in both untreated animals and those exposed to oxidative stress, and the effects of Tdp1 deficiency on tumor progression will be evaluated in mice that are predisposed to the development of premalignant prostate neoplasia. In addition, mice will be monitored for signs of accelerated aging. These studies are intended to clarify the mechanism by which Tdp1 deficiency leads to SCAN1, and may also help elucidate the role of oxidative stress in various other neurological disorders, cancer and aging.