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）和多种外源性物质（如多环芳香族化合物和氧化代谢物）自然发生。这些药物可以产生数百种不同的 DNA 损伤，包括氧化碱基损伤，氧化碱基损伤可以发生进一步的结构变化，从而产生更具毒性的 DNA 损伤。 如果这些 DNA 损伤得不到修复，它们就会改变基因组，导致细胞死亡，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 损伤。 自 1993 年以来，我们一直专注于碱基切除 DNA 修复途径，该途径可消除氧化性 DNA 损伤。 该途径由几个酶促步骤组成，包括识别氧化性 DNA 损伤、去除损伤以及将 DNA 序列恢复正常；我们的重点主要集中在第二步上。 在此步骤中，在 DNA 糖基化酶去除损伤碱基后，产生的脱嘌呤/脱嘧啶 (AP) 位点被 AP 核酸内切酶切割，以启动 DNA 修复合成。 AP 核酸内切酶有两个不同的家族，例如大肠杆菌核酸内切酶 IV (Endo IV/APN-1) 和核酸外切酶 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，我们鉴定并分离了编码 AP 核酸内切酶 APN-1 和 EXO-3 的基因。 随后我们对 APN-1 和 EXO-3 进行了纯化和功能表征。 这些研究揭示了 APN-1 的一些有趣的酶功能，这些功能不同于 EXO-3。 APN-1 具有四种酶活性来消除 DNA 损伤，而 EXO-3 仅具有其中两种活性。 因此，我们认为 APN-1 在消除秀丽隐杆线虫中的广泛 DNA 损伤方面比 EXO-3 重要得多。 此前，我们报道过，在缺乏 APN-1 的情况下，动物表现出高频率的突变，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损伤的根源是什么？ 在另一项研究中，我们最近发现线虫具有摄取转运蛋白 OCT-2，它可以使药物以及促氧化剂等有毒环境化合物进入动物体内。 我们进一步表明 oct-2 基因表达是由另一种转运蛋白 OCT-1 控制的。 在 OCT-1 缺失的情况下，oct-2 基因表达会受到刺激。 更引人注目的是，我们发现 OCT-2 上调会缩短动物的寿命并增加生殖细胞的死亡。 我们假设 OCT-2 介导从生长环境中摄取损伤 DNA 的基因毒性化合物，并且这些由此产生的 DNA 损伤由 APN-1 修复。 因此，我们的计划将提供新的见解，即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性。