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.

项目总结 碱性磷酸酶(AP)是细胞中最常见的DNA损伤之一，导致许多有害的 结果。许多环境毒素，包括溴酸钾和亚硝胺，都会导致DNA碱基 修饰，最终诱导AP位点的形成。AP部位有形成断链、阻塞的倾向 转录，并在复制过程中阻断DNA聚合酶。细胞必须有机制来 处理这些无处不在的损伤。在S相外形成的AP位点通过碱基切除修复 (BER)途径。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站点的第二条无错误路径 被发现涉及5-羟甲基胞嘧啶结合蛋白，胚胎干细胞特异性(HMCES)。 HMCES对人类健康很重要，在衰老和癌症中调节失调。HMCES包含SOS 反应相关多肽酶(SRAP)结构域，在生命的所有领域中都是保守的。HMCES SRAP结构域在单链DNA(SsDNA)中与AP位点形成独特的、稳定的DNA-蛋白质交联物(DPC)。 这种活动构成了一种新的DNA修复途径的基础，通过该途径，SRAP保护AP站点免受错误倾向的影响 复制过程中的聚合酶和核酸酶。然而，DPC稳定性的分子基础并不是 然而，它的特点很好。在与范德比尔特的Cortez实验室的合作下，确定了1.6？的晶体结构 在大肠杆菌SRAP蛋白YedK和含有AP位点的单链DNA之间形成的DPC。这个结构 揭示了一种蛋白质前所未有的天然活性，由此形成了高度稳定的噻唑烷键 在AP位点的开环形式和SRAP的不变N端Cys2残基之间。该结构 也表明SRAP对DNA结构具有特异性，这种结构会出现在停滞不前的复制分叉上。 这种结构将被用来破译形成和解决这一问题的化学和物理性质 通过探索SRAP-AP-DPC的结构和化学生物学，实现新的DNA-蛋白质化学连接 原子水平。目标1将阐明SRAP的交联机制，目标2将研究SRAP-DPC 在DNA复制结构的背景下形成。鉴于AP现场修复的重要性，以应对 环境毒素以及癌症和衰老，对SRAP AP位点交联的详细了解是 对于理解SRAP蛋白如何提供对人类健康至关重要的完全未知的途径至关重要。