The molecular basis of SRAP domain DNA-protein crosslinking

SRAP结构域DNA-蛋白质交联的分子基础

基本信息

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
项目类别：
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

项目摘要

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.

项目概要无碱基 (AP) 位点是细胞中最常见的 DNA 损伤之一，会导致多种疼痛许多环境毒素，包括溴酸钾和亚硝胺，都会导致 DNA 碱基的形成。修饰，最终诱导 AP 位点的形成 AP 位点表现出形成链断裂、阻断的倾向。转录并在复制过程中阻断 DNA 聚合酶，细胞必须具有这样的机制。通过碱基切除修复来处理这些在 S 期之外形成的普遍存在的损伤。 DNA 复制过程中遇到的 AP 位点被跨损伤 DNA 合成 (TLS) 绕过。最近，出现了第二种无差错的途径来处理与复制相关的 AP 位点。被发现涉及胚胎干细胞特异性蛋白 5-羟甲基胞嘧啶结合 (HMCES)。 HMCES 对人类健康很重要，并且在衰老和癌症中失调。响应相关肽酶 (SRAP) 结构域，在生命的所有领域中都是保守的。 SRAP 结构域与单链 DNA (ssDNA) 中的 AP 位点形成独特、稳定的 DNA-蛋白质交联 (DPC)。这一活性构成了一种新型 DNA 修复途径的基础，通过 SRAP 可以保护 AP 位点免受容易出错的影响然而，DPC 稳定性的分子基础并非如此。与范德比尔特大学 Cortez 实验室合作，确定了 1.6 Å 的晶体结构。大肠杆菌 SRAP 蛋白、YedK 和含有 AP 位点的 ssDNA 之间形成的 DPC。揭示了蛋白质前所未有的天然活性，从而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 端 Cys2 残基之间的关系。还表明 SRAP 对存在于停滞复制叉处的 DNA 结构具有特异性。该结构将用于破译该结构的形成和解析的化学和物理性质。通过探究 SRAP-AP-DPC 的结构和化学生物学，发现了新颖的 DNA-蛋白质化学连接目标 1 将阐明 SRAP 交联机制，目标 2 将研究 SRAP-DPC。鉴于 AP 位点修复对 DNA 复制结构的重要性。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经探索的途径至关重要。