The molecular basis of SRAP domain DNA-protein crosslinking

SRAP结构域DNA-蛋白质交联的分子基础

• 批准号: 10312706
• 负责人: Katherine Amidon Paulin
• 金额: $ 3.04万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312706
• 关键词: Active Sites; Aging; B-Cell Lymphomas; Base Excision Repairs; Binding; Binding Proteins; Biochemical; Biology; Bypass; Cells; Chemicals; Clinical; Collaborations; Critical Pathways; Crystallization; Cysteine; DNA; DNA Maintenance; DNA Repair; DNA Repair Pathway; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; DNA-protein crosslink; Data; Detergents; Down-Regulation; Enzymatic Biochemistry; Escherichia coli; Exhibits; Exposure to; Food; Food Additives; Foundations; Frequencies; Future; Genetic; Genetic Transcription; Health; Heavy Metals; Human; In Vitro; Kinetics; Knowledge; Lead; Lesion; Life; Malignant Neoplasms; Metabolism; Modeling; Modification; Molecular; Mutagenesis; Mutagens; Mutation; N-terminal; Nature; Nitrosamines; Orthologous Gene; Outcome; Oxidative Stress; Pathway interactions; Peptide Hydrolases; Play; Polymerase; Proliferating Cell Nuclear Antigen; Proteins; Reaction; Reader; Research; Resolution; Role; S Phase; SOS Response; Single-Stranded DNA; Site; Solvents; Specificity; Structure; Toxic Environmental Substances; Transact; UV Radiation Exposure; Virus Replication; Work; Zika Virus; base; crosslink; cytotoxicity; embryonic stem cell; genome integrity; in vivo; insight; irradiation; novel; nuclease; nucleobase; oxidative damage; pollutant; potassium bromate; repaired; response

PROJECT SUMMARY Abasic (AP) sites are one of the most common DNA lesions in cells and lead to numerous detrimental outcomes. Many environmental toxins, including potassium bromate and nitrosamines, lead to DNA base modification, ultimately inducing the formation of AP sites. AP sites exhibit tendency to form strand breaks, block transcription, and block DNA polymerases during replication. It is imperative for cells to have mechanisms to deal with these ubiquitous lesions. AP sites formed outside of S-phase are repaired via the base excision repair (BER) pathway. AP sites encountered during DNA replication are bypassed by translesion DNA synthesis (TLS) in an error-prone manner. Recently, a second, error-free pathway for processing replication-associated AP sites was discovered to involve the protein 5-hydroxymethlycytosine binding, embryonic stem cell-specific (HMCES). HMCES is important to human health and is dysregulated in aging and cancer. HMCES contains an SOS Response Associated Peptidase (SRAP) domain, which is conserved across all domains of life. The HMCES SRAP domain forms a unique, stable DNA-protein crosslink (DPC) to AP sites in single stranded DNA (ssDNA). This activity forms the basis of a novel DNA repair pathway whereby SRAP protects AP sites from error-prone polymerases and nucleases during replication. However, the molecular basis for the stability of the DPC is not yet well-characterized. In collaboration with the Cortez lab at Vanderbilt, a 1.6-Å crystal structure was determined of the DPC formed between the E. coli SRAP protein, YedK, and ssDNA containing an AP site. This structure reveals an unprecedented native activity of a protein whereby a highly stable thiazolidine linkage is formed between the ring-opened form of the AP site and the invariant N-terminal Cys2 residue of SRAP. The structure also suggests that SRAP has a specificity for DNA structures that would be present at stalled replication forks. This structure will be utilized to decipher the chemical and physical nature of formation and resolution of this novel DNA-protein chemical linkage by probing the structure and chemical biology of the SRAP-AP-DPC at the atomic level. Aim 1 will elucidate the SRAP crosslinking mechanism and Aim 2 will investigate SRAP-DPC formation in the context of DNA replication structures. Given the importance of AP site repair in response to environmental toxins as well as cancer and aging, a detailed understanding of SRAP AP site crosslinking is crucial for understanding how SRAP proteins provide a completely unexplored pathway critical for human health.

项目摘要 脱碱基（AP）位点是细胞中最常见的DNA损伤之一，并导致许多有害的DNA损伤。 结果。许多环境毒素，包括溴酸钾和亚硝胺，导致DNA碱基 修饰，最终诱导AP位点的形成。AP位点表现出形成链断裂、阻断 转录，并在复制过程中阻断DNA聚合酶。细胞必须有机制 处理这些无处不在的病变。在S期外形成的AP位点通过碱基切除修复 (BER)通路在DNA复制过程中遇到的AP位点被跨损伤DNA合成（TLS）绕过 以容易出错的方式。最近，用于处理与复制相关的AP站点的第二个无错误路径 被发现涉及蛋白质5-羟甲基胞嘧啶结合，胚胎干细胞特异性（HMCES）。 HMCES对人类健康很重要，在衰老和癌症中失调。HMCES包含SOS 反应相关肽酶（SRAP）结构域，在生命的所有领域都是保守的。HMCES SRAP结构域与单链DNA（ssDNA）中的AP位点形成独特的、稳定的DNA-蛋白质交联（DPC）。 这种活性形成了一种新的DNA修复途径的基础，SRAP通过这种途径保护AP位点不受易错蛋白的损伤。 聚合酶和核酸酶。然而，DPC稳定性的分子基础不是 但特征鲜明与范德比尔特的科尔特斯实验室合作，确定了1.6-ε晶体结构 E. coli SRAP蛋白、YedK和含有AP位点的ssDNA。这种结构 揭示了蛋白质前所未有的天然活性，由此形成高度稳定的噻唑烷键 AP位点的开环形式和SRAP的不变N-末端Cys 2残基之间。结构 还表明，SRAP具有特异性的DNA结构，将出现在停滞的复制叉。 这种结构将被用来破译化学和物理性质的形成和解决这一问题， 通过探测SRAP-AP-DPC的结构和化学生物学， 原子水平。目的1阐明SRAP交联机理，目的2研究SRAP-DPC 在DNA复制结构的背景下形成。鉴于AP部位修复在应对 环境毒素以及癌症和衰老，详细了解SRAP AP位点交联， 对于理解SRAP蛋白如何提供对人类健康至关重要的完全未探索的途径至关重要。