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: 10536876
• 负责人: Savannah Weeks Pollenz
• 金额: $ 4.34万
• 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/10536876
• 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; Foundations; 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; base; 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.

项目摘要/摘要 DNA经常被外源破坏，从暴露在紫外线下到体内的有毒化学物质 环境。为了修复这些因素造成的损害并保持基因组的稳定，细胞有多个 高效的DNA修复机制。然而，一些损害将不可避免地逃脱修复，如果损害的负担 太高了。未修复的DNA损伤会阻碍DNA合成，并对细胞造成严重后果 也是为了人类的健康。布朗等人的一项研究。以叠氮胸苷(AZT)为工具阻断E. Coli来发现解决复制分叉停滞的必要基因。AZT是一种胸苷类似物，可以 在合成过程中并入，防止引物延伸，导致复制停滞和单链 DNA缺口的形成。两个基因yoaA和HOLC被发现对解决停滞的DNA复制至关重要 在AZT处理的大肠杆菌细胞中。YoaA基因编码一个类似XPD/Rad3的解旋酶。四个人类XPD/Rad- 3类解旋酶(FANCJ、XPD、RTEL1和CHLR1)有助于基因组稳定，如果受到损害，可以 会导致各种遗传病并增加患癌症的风险。HOLC基因编码chi，chi是一种 是两个不同建筑群的一部分。CHI是DNA聚合酶III钳位加载器的一个附属亚单位，它形成 含有全酶的复合体。CHI还与YoaA结合，以产生一个功能性的YoaA-chi解旋酶。池是众所周知的 结合单链DNA结合蛋白(SSB)，这种相互作用对于解决停滞不前的损伤是必要的 复制。SSB是一种必需的蛋白质，存在于生命的各个领域，覆盖单链(Ss)DNA，以及 与十几种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害是未知的。因此，这个奖学金的目的是描述SSB 用生化技术与YoaA-chi相互作用以了解这一新的修复途径。它是 假设的SSB调节YoaA-chi解开双链DNA的能力，以解决 基于初步数据的复制分叉，显示YoaA-chi的解旋酶活性在 SSB的存在。SSB如何与YoaA-chi结合将被阐明，无论是通过chi上的已知位置还是 通过可能在YoaA(目标1)上的新的互动。因为SSB调节多种DNA结合蛋白 通过不同的机制，我们将研究SSB可以调控的YoaA-chi的几个方面。会是 确定SSB是否改变了YoaA-chi的底物亲和力(Aim 2)或YoaA-chi的解旋酶活性(Aim 3)。这将是关于SSB如何调控YoaA-chi以及这些蛋白质在 新的DNA修复机制。这项研究也将对我的培训做出重大贡献，成为 佛罗里达大学独立的生物化学家和环境学家将使我取得成功。