Repair Mechanisms For Oxidative DNA Damage

DNA 氧化损伤的修复机制

• 批准号: 9147336
• 负责人: David Wilson
• 金额: $ 39.35万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9147336
• 关键词: Aging; Aging-Related Process; Alkylation; Back; Base Excision Repairs; Binding; Biochemical; Cell Death; Cell Line; Cell Respiration; Cells; Chromosomal Stability; Cockayne Syndrome; Complement component C1; Complex; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA glycosylase; Disease; Disease susceptibility; Evolution; Excision; Exhibits; Family member; Flap Endonucleases; Foundations; Functional disorder; Genetic Transcription; Genome; Human; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Individual; Lead; Lesion; Ligase; Link; Lipids; Location; Lyase; Maintenance; Mitochondria; Modification; Molecular; Molecular Conformation; Mutate; NPM1 gene; Nature; Nuclear; Nucleic Acids; Nucleosomes; Nucleotides; Organism; Oxidation-Reduction; Pathway interactions; Play; Polymerase; Population; Positioning Attribute; Post-Translational Protein Processing; Premature aging syndrome; Process; Proteins; RECQL4 gene; RECQL5 gene; RNA; Reactive Oxygen Species; Relative (related person); Repair Complex; Reporting; Research; Role; Rothmund-Thomson syndrome; Scaffolding Protein; Series; Single Strand Break Repair; Site; Surface; Surgical incisions; System; Toxic Environmental Substances; Uracil; Variant; Vertebral column; Work; age related; base; cell growth; chromatin remodeling; coping; crosslink; cytotoxic; disorder risk; endonuclease; genetically modified cells; helicase; inorganic phosphate; novel; nuclease; nucleophosmin; oxidative DNA damage; phosphodiester; repair enzyme; repaired; response; seal; tumor; uracil-DNA glycosylase

Using molecular, biochemical and structural approaches, we have broadly contributed to defining how specific human BER proteins recognize and process target lesions, as well as coordinate with other components of the protective system. The research has centered largely on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian protein for repairing abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a non-enzymatic scaffold protein that facilitates the efficient execution of single-strand break (SSB) repair. Some of the key findings during the course of the project include: (i) in addition to abasic sites in conventional duplex DNA, APE1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and APE1 can endonucleolytically destroy damaged RNA; (ii) APE1 contributes to the repair of 3-modifications in DNA, such as mismatches, phosphate groups, phosphogycolates and tyrosyl residues; (iii) the DNA repair function of APE1 is regulated in part by post-translational modification, such as S-glutathionylation; (iv) inhibition of APE1 is a potential mechanism for the genotoxic and co-carcinogenic effects of lead, an important environmental toxin; (v) APE1 communicates with CSB, a protein defective in the premature aging disorder, Cockayne syndrome; (vi) XRCC1 directly associates with the replication/repair protein, PCNA, establishing a novel link between the DNA repair machinery and replication factories; (vii) XRCC1 coordinates disparate responses and multi-protein repair complexes that are dependent on the nature and context of the DNA damage; (viii) the different regions of XRCC1 play distinct roles in coordinating repair complex assembly, and the population variant Arg280His exhibits reduced stability at DNA damage foci, suggesting that it may represent a disease susceptibility factor; (ix) XRCC1 supports an emerging pathway for uracil repair, termed replication-associated BER, through a physical association with UNG2, the major nuclear uracil DNA glycosylase; (x) the interaction of XRCC1 with the DNA repair enzyme PNKP functions to retain XRCC1 at DNA damage sites and to promote repair of alkylation damage; (xi) the flap-endonuclease FEN1 plays a role in repairing mitochondrial oxidative DNA damage through a long-patch BER pathway; (xii) RECQL4, a human RecQ helicase mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome, regulates BER capacity both directly and indirectly; and (xiii) RECQL5, another RECQ helicase family member, modulates and/or directly participates in BER of endogenous DNA damage, thereby promoting chromosome stability in normal human cells. Our most recent work has demonstrated that (i) the DNA glycosylase NEIL1 recognizes specifically ICLs in DNA, and can obstruct the efficient removal of these lethal lesions; (ii) the multifunctional protein nucleophosmin (a.k.a., NPM1) is a modulator of BER capacity by controlling BER protein levels and regulating the nucleolar localization of several BER enzymes; (iii) the efficiency of APE1 binding and strand incision are influenced by the domain sequence, conformation and AP site location/relative positioning within telomeric and CAG/CTG repeat sequences; and (iv) specific APE1 variants (e.g., the tumor-associated R237C variant) are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site, suggesting the evolution of distinct surface residues that permit efficient processing of abasic sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms. Currently, a main focus is to establish genetically modified cell lines to dissect out the precise contribution of each proposed function of APE1 (i.e. its nuclease activity, redox regulatory role, etc.) in cell growth/viability, genome maintenance, and protection against DNA-damaging agents. Defining which of the many reported functions of APE1 are critical to normal cellular activity is a key step towards understanding the potential relationship of the protein to the aging process and disease risk.

利用分子、生化和结构方法，我们在定义特定人类 BER 蛋白如何识别和处理目标病变以及与保护系统的其他组成部分协调方面做出了广泛贡献。 该研究主要集中在无嘌呤/无嘧啶核酸内切酶 1 (APE1)（用于修复 DNA 脱碱基位点的主要哺乳动物蛋白）和 X 射线交叉互补 1 (XRCC1)（一种非酶支架蛋白，有助于有效执行单链断裂（SSB）修复。 该项目过程中的一些关键发现包括：(i) 除了传统双链 DNA 中的脱碱基位点之外，APE1 还能够切割 DNA 复制、转录和类别转换重组过程中形成的 DNA 构象中的 AP 位点， APE1可以通过核酸内切方式破坏受损的RNA； (ii) APE1 有助于修复 DNA 中的 3-修饰，例如错配、磷酸基团、磷酸乙二醇酯和酪氨酰残基； (iii) APE1的DNA修复功能部分受到翻译后修饰的调节，例如S-谷胱甘肽化； (iv) 抑制 APE1 是铅（一种重要的环境毒素）的遗传毒性和协同致癌作用的潜在机制； (v) APE1 与 CSB 通讯，CSB 是一种在早衰症、科凯恩综合征中存在缺陷的蛋白质； (vi) XRCC1 直接与复制/修复蛋白 PCNA 结合，在 DNA 修复机制和复制工厂之间建立新的联系； (vii) XRCC1 协调取决于 DNA 损伤的性质和背景的不同反应和多蛋白修复复合物； (viii) XRCC1的不同区域在协调修复复合物组装中发挥不同的作用，群体变体Arg280His在DNA损伤灶处表现出稳定性降低，表明它可能代表一种疾病易感因素； (ix) XRCC1 通过与主要核尿嘧啶 DNA 糖基化酶 UNG2 的物理关联，支持一种新兴的尿嘧啶修复途径，称为复制相关 BER； (x) XRCC1与DNA修复酶PNKP的相互作用起到将XRCC1保留在DNA损伤位点并促进烷基化损伤修复的作用； (xi) 瓣核酸内切酶 FEN1 通过长补丁 BER 途径在修复线粒体氧化 DNA 损伤中发挥作用； (xii) RECQL4 是一种人类 RecQ 解旋酶，在大约三分之二的 Rothmund-Thomson 综合征患者中发生突变，直接和间接调节 BER 能力； (xiii)RECQL5，另一个RECQ解旋酶家族成员，调节和/或直接参与内源DNA损伤的BER，从而促进正常人类细胞中的染色体稳定性。 我们最近的工作表明，(i) DN​​A 糖基化酶 NEIL1 可以特异性识别 DNA 中的 ICL，并且可以阻碍这些致命病变的有效去除； (ii) 多功能蛋白核磷蛋白（又名 NPM1）是 BER 能力的调节剂，通过控制 BER 蛋白水平和调节几种 BER 酶的核仁定位； (iii) APE1结合和链切割的效率受到结构域序列、构象和AP位点位置/端粒和CAG/CTG重复序列内的相对定位的影响； (iv) 特定的 APE1 变体（例如肿瘤相关的 R237C 变体）对 AP 位点附近的核小体复合物特别敏感，这表明不同表面残基的进化允许在蛋白质背景下有效加工脱碱基位点-DNA复合物独立于经典染色质重塑机制。 目前，主要焦点是建立转基因细胞系，以剖析 APE1 的每个拟议功能（即其核酸酶活性、氧化还原调节作用等）在细胞生长/活力、基因组维护和 DNA 保护中的精确贡献- 破坏剂。 确定 APE1 的众多已报道功能中的哪些功能对正常细胞活动至关重要，是了解该蛋白质与衰老过程和疾病风险之间潜在关系的关键一步。

项目成果

Characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1.

• DOI: 10.1016/j.jmb.2011.06.050
• 发表时间: 2011-09-02
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Kim WC;Berquist BR;Chohan M;Uy C;Wilson DM 3rd;Lee CH
• 通讯作者: Lee CH

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

• DOI: 10.1016/j.dnarep.2010.04.002
• 发表时间: 2010-07-01
• 期刊: DNA repair
• 影响因子: 3.8
• 作者: Akbari M;Solvang-Garten K;Hanssen-Bauer A;Lieske NV;Pettersen HS;Pettersen GK;Wilson DM 3rd;Krokan HE;Otterlei M
• 通讯作者: Otterlei M

Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins.

核磷蛋白调节碱基切除修复蛋白的稳定性、活性和核仁积累。

• DOI: 10.1091/mbc.e13-12-0717
• 发表时间: 2014
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Poletto,Mattia;Lirussi,Lisa;Wilson3rd,DavidM;Tell,Gianluca
• 通讯作者: Tell,Gianluca

A novel link to base excision repair?

• DOI: 10.1016/j.tibs.2010.01.003
• 发表时间: 2010-05
• 期刊: TRENDS IN BIOCHEMICAL SCIENCES
• 影响因子: 13.8
• 作者: Wilson, David M., III;Seidman, Michael M.
• 通讯作者: Seidman, Michael M.

The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1.

• DOI: 10.1016/j.dnarep.2012.01.001
• 发表时间: 2012-04-01
• 期刊: DNA REPAIR
• 影响因子: 3.8
• 作者: Hanssen-Bauer, Audun;Solvang-Garten, Karin;Gilljam, Karin Margaretha;Torseth, Kathrin;Wilson, David M., III;Akbari, Mansour;Otterlei, Marit
• 通讯作者: Otterlei, Marit