Preferential single-strand break repair in the active genes of mammalian cells

描述（由申请人提供）：在各种神经系统疾病的发展中基因/环境相互作用已被充分记录。DNA损伤，包括单链断裂（SSBs），就是这种相互作用的结果。DNA SSB修复（SSBR）缺陷可能会对人类健康造成严重影响。几种神经系统疾病已经被鉴定和表征，这是由于缺乏DNA末端加工活性，由酶如aprataxin和TDP 1催化。人多核苷酸激酶3 '-磷酸酶（PNKP）是另一种SSBR酶，可加工内源性和外源性遗传毒性剂产生的3'-P和5 '-OH末端。这些DNA末端需要被处理以恢复基因组的完整性，因为未修复的SSB会阻止转录，这对所有细胞都是有害的。我们假设活性基因转录链中的SSB优先通过SSBR的子途径修复，我们称之为转录偶联SSBR（TC-SSBR），并且PNKP在转录序列中的3 '-P和5'-OH末端加工中起着至关重要的作用。我们现在已经发现PNKP存在于线粒体中。PNKP与核和线粒体RNA聚合酶的关联，以及PNKP与转录基因的优先关联，进一步支持了我们的优先修复活跃转录基因的假设。我们对PNKP与Ataxin-3（ATXN 3）（一种负责脊髓小脑共济失调3型（也称为Machado-Joseph病（MJD/SCA 3））的蛋白质）的关联的惊人观察促使我们研究PNKP在疾病发病机制中的作用。MJD/SCA 3是由ATXN 3基因编码区CAG重复序列（poly-Q）扩增引起的致死性常染色体显性遗传病。没有治疗这种疾病的方法。Poly-Q疾病的一个共同病理特征是核内包涵体的积累。然而，致病性ATXN 3（ATXN 3-Q72）引起神经变性的机制仍然不清楚。我们的初步数据表明，ATXN 3的病理形式在体外阻断了PNKP介导的SSBR活性。因此，病理形式也可能阻断PNKP介导的TC-SSBR。神经系统会遇到高水平的氧化应激，消耗约20%的吸入氧气。此外，有丝分裂后神经元具有高转录率，这可能进一步增加这些细胞对TC-SSBR的依赖性，以维持核和mt基因组的完整性。因此，为了了解SSBR的分子生物学和疾病过程，我们的项目将有三个具体的目的，以验证假设：1。ATXN 3-Q72阻断PNKP介导的核TC-SSBR; 2. ATXN 3-Q72阻断PNKP介导的mtTC-SSBR;和3. PNKP的异位表达将拯救ATXN 3-Q72介导的细胞毒性。我们的长期目标是确定共济失调发展的机制基础，并制定预防或治疗人群中MJD/SCA 3的新策略。 公共卫生相关性：Machado-Joseph病，或脊髓小脑共济失调3型（MJD/SCA 3），是世界上最常见的显性遗传性共济失调;然而，由于对该疾病的分子机制尚不清楚，因此没有可用的治疗方法。我们的研究旨在确定一个新的优先修复转录基因中单链DNA断裂的子途径，并表明该途径的缺陷可能在SCA 3/MJD中起因果作用。我们的预期结果最终将导致新的治疗干预策略，甚至是预防与DNA损伤和修复有关的疾病的发病的方法。

DESCRIPTION (provided by applicant): Gene/environment interactions in the development of various neurological diseases have been well documented. DNA damage, including single-strand breaks (SSBs), is the outcome of one such interaction. Defects in DNA SSB repair (SSBR) may have striking human health consequences. Several neurological diseases have already been identified and characterized that are due to the lack of DNA end-processing activities, catalyzed by enzymes such as aprataxin and TDP1. Human polynucleotide kinase 3'-phosphatase (PNKP) is another SSBR enzyme that processes 3'-P and 5'-OH ends, generated both endogenously and by exogenous genotoxic agents. These DNA termini need to be processed to restore genomic integrity, because unrepaired SSBs would block transcription, which is detrimental in all cells. We hypothesize that SSBs in the transcribed strand of active genes are preferentially repaired via a subpathway of SSBR, which we call transcription-coupled SSBR (TC-SSBR), and that PNKP plays a vital role for 3'-P and 5'-OH end- processing in the transcribed sequences. We have now found that PNKP is present in the mitochondria. The association of PNKP with nuclear and mt RNA polymerases, and preferential association of PNKP with transcribed genes, further supports our hypothesis of preferential repair of actively transcribed genes. Our surprising observation of the association of PNKP with Ataxin-3 (ATXN3), a protein responsible for spinocerebellar ataxia type 3, also called Machado-Joseph Disease (MJD/SCA3), prompted us to investigate PNKP's role in the pathogenesis of the disease. MJD/SCA3 is a fatal, autosomal dominant disorder caused by CAG repeat (poly-Q) expansion in the coding region of the ATXN3 gene. There is no therapy available for this disease. A common pathological feature of poly-Q diseases is the accumulation of intranuclear inclusions. However, the mechanism by which pathogenic ATXN3 (ATXN3-Q72) causes neurodegeneration is still not clearly understood. Our preliminary data showed that the pathological form of ATXN3 blocked PNKP-mediated SSBR activities in vitro. It is thus likely that the pathological form will block PNKP-mediated TC-SSBR as well. The nervous system encounters a high level of oxidative stress, consuming ~20% of inhaled oxygen. Additionally, postmitotic neurons have a high transcriptional rate, which might further increase the dependency of these cells on TC-SSBR to maintain the integrity of both the nuclear and mt genomes. Therefore, to understand the molecular biology of SSBR and the disease process, our project will have three Specific Aims, to test the hypotheses that: 1. ATXN3-Q72 blocks PNKP-mediated nuclear TC-SSBR; 2. ATXN3-Q72 blocks PNKP-mediated mtTC-SSBR; and 3. Ectopic expression of PNKP will rescue ATXN3-Q72-mediated cellular toxicity. Our long-term goal is to determine the mechanistic basis for the development of Ataxia and to develop new strategies for the prevention or treatment of MJD/SCA3 in the human population. PUBLIC HEALTH RELEVANCE: Machado-Joseph disease, or spinocerebellar ataxia type 3 (MJD/SCA3), is the most common dominantly inherited ataxia worldwide; however, no therapy is available because the molecular mechanism responsible for the disease is not clearly understood. Our study is aimed at identifying a new subpathway for preferential repair of single-strand DNA breaks in the transcribed genes, and showing that deficiencies in this pathway could play a causal role in SCA3/MJD. Our expected results should ultimately lead to new therapeutic intervention strategies, or even to approaches for preventing the onset of diseases etiologically linked to DNA damage and repair.