How SSB Regulates YoaA-chi's Function in DNA Damage Repair

SSB 如何调节 YoaA-chi 的 DNA 损伤修复功能

• 批准号: 10684693
• 负责人: Savannah Weeks Pollenz
• 金额: $ 4.43万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2025-08-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684693
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Affinity; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Models; Bypass; Cell Cycle Arrest; Cell Death; Cell Survival; Cells; Closure by clamp; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Polymerase III; DNA Repair; DNA Repair Enzymes; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Binding Proteins; Data; Disease; Dissociation; Environment; Equilibrium; Escherichia coli; Essential Genes; Exogenous Factors; Exposure to; Fanconi' s Anemia; Fellowship; Florida; Fluorescence Resonance Energy Transfer; Genes; Genetic; Genetic Diseases; Genetic Recombination; Genome Stability; Genomic Instability; Goals; Health; Holoenzymes; Human; In Vitro; Lead; Left; Lesion; Life; Location; Malignant Neoplasms; Measures; Mediating; Mutation; Nucleotide Excision Repair; Pathway interactions; Patients; Poison; Predisposition; Primer Extension; Proteins; Research; SS DNA BP; Single-Stranded DNA; Site; Source; Stretching; Techniques; Thymidine; Toxin; Training; Ultraviolet Rays; Universities; Work; Xeroderma Pigmentosum; Zidovudine; analog; cancer risk; ds-DNA; helicase; insight; mutant; novel; paralogous gene; pollutant; prevent; recruit; repaired; therapy development; tool

Project Summary/Abstract DNA is frequently damaged by exogenous sources ranging from exposure to UV light to toxic chemicals in the environment. To fix the damage caused by these agents and maintain genomic stability, cells have multiple efficient DNA repair mechanisms. Some damage, though, will inevitably escape repair if the burden of damage is too high. Unrepaired DNA damage can block DNA synthesis and have serious consequences for the cell and for human health. A study by Brown et al. used azidothymidine (AZT) as a tool to block replication in E. coli to discover essential genes for resolving stalled replication forks. AZT is a thymidine analog that can be incorporated during synthesis and prevents primer extension, causing replication to stall and single-strand DNA gaps to form. Two genes, yoaA and holC, were discovered to be vital for resolving stalled DNA replication in AZT treated E. coli cells. The yoaA gene encodes for an XPD/Rad3-like helicase. The four human XPD/Rad- 3 like helicases (FANCJ, XPD, RTEL1, and CHLR1) contribute to genomic stability and if compromised, can cause various genetic diseases and an increased risk of cancer. The holC gene encodes for chi, which is a part of two different complexes. Chi is an accessory subunit of the DNA polymerase III clamp loader and forms a complex with the holoenzyme. Chi also binds YoaA to create a functional YoaA-chi helicase. Chi is known to bind single-stranded DNA binding protein (SSB) and this interaction is necessary for resolving lesions that stall replication. SSB is an essential protein found in all domains of life, coats single-stranded (ss) DNA, and interacts with over a dozen DNA repair and replication proteins. How YoaA, chi, and SSB work together to resolve damage that halts replication is unknown. Therefore, this fellowship aims to characterize SSB interactions with YoaA-chi with biochemical techniques to understand this novel repair pathway. It is hypothesized SSB regulates the ability of YoaA-chi to unwind double-stranded DNA to resolve lesions at the replication fork based on preliminary data which shows that the helicase activity of YoaA-chi is decreased in the presence of SSB. How SSB binds YoaA-chi will be elucidated, be it either by the known location on chi or by a new interaction possibly on YoaA (aim 1). Because SSB regulates a variety of DNA-binding proteins through various mechanisms, several facets of YoaA-chi that SSB could regulate will be investigated. It will be determined if SSB changes the substrate affinity of YoaA-chi (aim 2) or the helicase activity of YoaA-chi (aim 3). This will be the first study into how SSB regulates YoaA-chi and the contribution these proteins have in a novel DNA repair mechanism. This research will also provide significant contributions in my training to become an independent biochemist and the environment at the University of Florida will allow me to be successful.

项目概要/摘要 DNA 经常受到外源性损伤，包括暴露于紫外线和环境中的有毒化学物质。 环境。为了修复这些物质造成的损伤并维持基因组稳定性，细胞具有多种 有效的DNA修复机制。然而，如果损坏负担过重，有些损坏将不可避免地无法修复 太高了。未修复的 DNA 损伤会阻碍 DNA 合成并对细胞产生严重后果 以及为了人类的健康。布朗等人的一项研究。使用叠氮胸苷（AZT）作为阻断大肠杆菌复制的工具。 大肠杆菌发现解决停滞复制叉的必需基因。 AZT 是一种胸苷类似物，可以 在合成过程中掺入并阻止引物延伸，导致复制停滞和单链 DNA缺口形成。两个基因 yoaA 和 holC 被发现对于解决 DNA 复制停滞问题至关重要 在 AZT 处理的大肠杆菌细胞中。 yoaA 基因编码 XPD/Rad3 样解旋酶。四个人类 XPD/Rad- 3 个类似的解旋酶（FANCJ、XPD、RTEL1 和 CHLR1）有助于基因组稳定性，如果受到损害，可以 导致各种遗传疾病并增加患癌症的风险。 holC 基因编码 chi，它是 两个不同复合体的一部分。 Chi 是 DNA 聚合酶 III 钳加载器的辅助亚基，并形成 与全酶的复合物。 Chi 还结合 YoaA 以创建功能性 YoaA-chi 解旋酶。众所周知，Chi 结合单链 DNA 结合蛋白 (SSB)，这种相互作用对于解决停滞的损伤是必要的 复制。 SSB 是生命所有领域中发现的一种必需蛋白质，覆盖单链 (ss) DNA，并且 与十多种 DNA 修复和复制蛋白相互作用。 YoaA、chi 和 SSB 如何协同工作 解决导致复制停止的损坏尚不清楚。因此，本研究金旨在表征 SSB 通过生化技术与 YoaA-chi 相互作用，以了解这种新的修复途径。这是 假设 SSB 调节 YoaA-chi 解开双链 DNA 的能力，以解决损伤 复制叉基于初步数据，表明 YoaA-chi 的解旋酶活性在 单边带的存在。 SSB 如何结合 YoaA-chi 将被阐明，无论是通过 chi 上的已知位置还是通过 通过可能在 YoaA 上的新交互（目标 1）。因为 SSB 调节多种 DNA 结合蛋白 通过各种机制，SSB 可以调节的 YoaA-chi 的几个方面将得到研究。这将是 确定 SSB 是否改变 YoaA-chi 的底物亲和力（目标 2）或 YoaA-chi 的解旋酶活性（目标 3）。这将是第一个研究 SSB 如何调节 YoaA-chi 以及这些蛋白质在 新颖的DNA修复机制。这项研究也将为我的培训做出重大贡献，使我成为 一名独立的生物化学家和佛罗里达大学的环境将使我取得成功。