Role of C. elegans AP endonuclease, APN-1, in removing DNA lesions generated through transporter-mediated uptake of naturally occurring toxic compounds.

线虫 AP 核酸内切酶 (APN-1) 在消除转运蛋白介导的天然有毒化合物摄取产生的 DNA 损伤中的作用。

• 批准号: RGPIN-2017-04244
• 负责人: Ramotar, Dindial
• 金额: $ 2.48万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662970
• 关键词: Role; C.; elegans; AP; endonuclease

Damage to the DNA can occur naturally by endogenous agents such as reactive oxygen species, and by several exogenous agents such as polycyclic aromatic compounds and oxidized metabolites. These agents can create hundreds of distinct DNA lesions including oxidative base damage that can undergo further structural changes to give rise to even more toxic DNA lesions. If these DNA lesions are not repaired, they can alter the genome to cause cell death and thus lead to many age related diseases. To avoid these deleterious effects, there are several pathways that act to remove specific classes of DNA lesions. Since 1993, we have been focusing on the base-excision DNA repair pathway that acts to remove oxidative DNA lesions. This pathway is made up of several enzymatic steps involving the recognition of the oxidative DNA lesions, removing the lesions, and restoring the DNA sequence to normal; our focus was mostly on the second step. In this step, following removal of the damage base by DNA glycosylase, the resulting apurinic/apyrimidinic (AP) site is cleaved by an AP endonuclease to initiate DNA repair synthesis. There are two distinct families of AP endonucleases, exemplified by Escherichia coli endonuclease IV (Endo IV/APN-1) and exonuclease III (Exo-3). Both family members are conserved in the yeast Saccharomyces cerevisiae as well as in the worm Caenorhabditis elegans. In C. elegans, we have identified and isolated the genes encoding the AP endonucleases, APN-1 and EXO-3. We subsequently purified and functionally characterized both APN-1 and EXO-3. These studies revealed some interesting enzymatic functions of APN-1 that are distinct from EXO-3. While APN-1 possesses four enzymatic activities to remove DNA lesions, EXO-3 has only two of the activities. Therefore, we believe APN-1 is far more important than EXO-3 to remove a wide range of DNA lesions in C. elegans. Previously, we reported that in the absence of APN-1 the animals exhibited a high frequency of mutations, suggesting that APN-1 is required to repair damaged DNA to prevent genetic instability. So what is the source of the DNA lesions? In a separate study, we recently discovered that C. elegans has an uptake transporter OCT-2, which can allow the entry of pharmaceutical drugs as well as toxic environmental compounds such as pro-oxidants into the animals. We further showed that oct-2 gene expression is controlled by another transporter OCT-1. In the absence of OCT-1, oct-2 gene expression is stimulated. More strikingly, we found that OCT-2 upregulation shortened the lifespan and increased germ cell death of the animals. We hypothesize that OCT-2 mediates the uptake from the growth environment genotoxic compounds that damage the DNA, and these resulting DNA lesions are repaired by APN-1. Our program, therefore, will provide novel insights how active uptake transporters can induce genetic instability in cells compromised for DNA repair.

内源性剂（例如活性氧）以及几种外源性剂，例如多环芳族化合物和氧化代谢产物，对DNA的损害可能自然发生。这些药物可以产生数百种不同的DNA病变，包括氧化碱损伤，这些损伤可能会发生进一步的结构变化，从而导致更具毒性的DNA病变。 如果未修复这些DNA病变，它们可以改变基因组以引起细胞死亡，从而导致许多与年龄相关的疾病。 为了避免这些有害影响，有几种途径可以消除特定类别的DNA病变。 自1993年以来，我们一直专注于可去除氧化DNA病变的基础拆卸DNA修复途径。 该途径由几个酶促步骤组成，涉及识别氧化DNA病变，去除病变并将DNA序列恢复到正常状态。我们的重点主要是第二步。 在此步骤中，在通过DNA糖基化酶去除损伤碱后，由AP核酸内切酶切割所得的肾上腺素/apyrimidinic（AP）位点以启动DNA修复合成。 有两个不同的AP核酸内切酶家族，例如大肠杆菌核酸内切酶IV（Endo IV/APN-1）和外切核酸酶III（EXO-3）。 这两个家庭成员都在酿酒酵母的酵母糖疗法以及烈性虫的蠕虫炎。 在秀丽隐杆线虫中，我们鉴定并隔离了编码AP核酸内切酶APN-1和EXO-3的基因。 随后，我们纯化并在功能上表征了APN-1和EXO-3。 这些研究揭示了APN-1的一些有趣的酶促功能，这些功能与EXO-3不同。 尽管APN-1具有四种酶促活性以去除DNA病变，但Exo-3只有两种活性。 因此，我们认为APN-1比EXO-3重要得多，可以去除秀丽隐杆线虫中的广泛的DNA病变。 以前，我们报道说，在没有APN-1的情况下，动物表现出很高的突变频率，这表明需要APN-1来修复受损的DNA以防止遗传不稳定。 那么DNA病变的来源是什么？ 在另一项研究中，我们最近发现秀丽隐杆线虫具有摄取转运蛋白OCT-2，该转运蛋白可以允许进入药物以及有毒环境化合物，例如促氧化剂进入动物。 我们进一步表明，OCT-2基因表达受到另一个转运蛋白OCT-1的控制。 在没有OCT-1的情况下，刺激了OCT-2基因表达。 更引人注目的是，我们发现OCT-2上调缩短了寿命并增加了动物的生殖细胞死亡。 我们假设OCT-2介导了损害DNA的生长环境遗传毒性化合物的摄取，并且这些由此产生的DNA病变通过APN-1修复。 因此，我们的程序将提供新颖的见解，即主动摄取转运蛋白如何诱导损害DNA修复的细胞中的遗传不稳定性。