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.

DNA损伤可由内源性物质（如活性氧）和几种外源性物质（如多环芳香族化合物和氧化代谢物）自然发生。这些药物可以造成数百种不同的DNA损伤，包括氧化性碱基损伤，这种损伤可以经历进一步的结构变化，从而产生更多的毒性DNA损伤。如果这些DNA损伤得不到修复，它们会改变基因组，导致细胞死亡，从而导致许多与年龄有关的疾病。为了避免这些有害影响，有几种途径可以去除特定类型的DNA损伤。自1993年以来，我们一直专注于碱基切除DNA修复途径，其作用是去除氧化性DNA损伤。该途径由几个酶促步骤组成，包括识别氧化DNA损伤，去除损伤，并恢复DNA序列正常；我们主要关注的是第二步。在这一步中，在DNA糖基化酶去除损伤碱基后，产生的无尿嘧啶/无嘧啶（AP）位点被AP内切酶切割，以启动DNA修复合成。AP内切酶有两个不同的家族，如大肠杆菌内切酶IV （Endo IV/APN-1）和外切酶III （Exo-3）。这两个家族成员在酿酒酵母和秀丽隐杆线虫中都是保守的。在秀丽隐杆线虫中，我们已经鉴定并分离了AP内切酶APN-1和EXO-3的编码基因。我们随后对APN-1和EXO-3进行了纯化和功能表征。这些研究揭示了APN-1与EXO-3不同的一些有趣的酶功能。APN-1具有四种去除DNA损伤的酶活性，而EXO-3只有两种活性。因此，我们认为APN-1在清除秀丽隐杆线虫中广泛的DNA损伤方面远比EXO-3重要。先前，我们报道了在APN-1缺失的情况下，动物表现出高频率的突变，这表明APN-1是修复受损DNA以防止遗传不稳定所必需的。那么DNA损伤的来源是什么呢？在另一项研究中，我们最近发现秀丽隐杆线虫有一种摄取转运体OCT-2，它可以允许药物以及有毒的环境化合物（如促氧化剂）进入动物体内。我们进一步发现oct-2基因的表达受另一个转运体OCT-1的控制。在OCT-1缺失的情况下，oct-2基因表达受到刺激。更令人惊讶的是，我们发现OCT-2的上调缩短了动物的寿命，增加了生殖细胞的死亡。我们假设OCT-2介导从生长环境中摄取损害DNA的基因毒性化合物，这些DNA损伤由APN-1修复。因此，我们的项目将提供新的见解，主动摄取转运蛋白如何在DNA修复受损的细胞中诱导遗传不稳定。