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 Abasic（AP）位点是细胞中最常见的DNA病变之一，导致许多有害 结果。许多环境毒素，包括溴酸钾和亚硝胺，导致DNA碱 修改，最终诱导AP位点的形成。 AP站点提取形成链断裂的趋势，阻塞 转录和在复制过程中阻断DNA聚合酶。必须有机制的机制 处理这些普遍存在的病变。 S相外形成的AP站点通过基本惊喜维修修复 （BER）途径。在DNA复制过程中遇到的AP位点通过Translesion DNA合成（TLS）绕过 以错误的方式。最近，用于处理复制相关的AP位点的第二个无错误途径 被发现涉及蛋白质5-羟甲环霉素结合，胚胎干细胞特异性（HMCE）。 HMCE对人类健康很重要，并且在衰老和癌症中失调。 HMCES包含一个SOS 响应相关的肽酶（SRAP）结构域，该结构域在生命的所有领域中都保守。 hmces SRAP结构域形成了独特的，稳定的DNA蛋白交联（DPC），可在单个链DNA（ssDNA）中的AP位点。 该活动构成了新型DNA修复途径的基础，从而使SRAP保护AP位点免受易用错误 复制过程中的聚合酶和核素。但是，DPC稳定性的分子基础不是 尚未表征。与Vanderbilt的Cortez Lab合作，确定了1.6-Å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。这个结构 揭示了一种蛋白质的前所未有的天然活性，从而形成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住宅之间。结构 还表明，SRAP具有对DNA结构的特异性，而DNA结构将存在于停滞的复制叉处。 这种结构将被用来破译形成和解决方案的化学和物理性质 通过探测SRAP-AP-DPC的结构和化学生物学的新型DNA蛋白化学连接 原子水平。 AIM 1将阐明SRAP交联机制，AIM 2将研究SRAP-DPC 在DNA复制结构的背景下形成。考虑到AP站点维修的重要性 环境毒素以及癌症和衰老，对SRAP AP网站交联的详细了解是 对于理解SRAP蛋白如何为人类健康至关重要的完全出乎意料的途径至关重要。