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.

项目总结/摘要 DNA经常受到外源性来源的损伤，从暴露于紫外线到细胞中的有毒化学物质， 环境为了修复由这些试剂引起的损伤并维持基因组稳定性，细胞具有多个 有效的DNA修复机制然而，如果损害的负担 太高了未修复的DNA损伤会阻碍DNA合成，对细胞造成严重后果 和人类健康。Brown等人的一项研究使用叠氮胸苷（AZT）作为阻断E. 发现解决停滞复制叉的必需基因。AZT是一种胸苷类似物， 在合成过程中掺入，并防止引物延伸，导致复制停止和单链 DNA缺口形成。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E.大肠杆菌细胞。yoaA基因编码XPD/Rad 3样解旋酶。四个人类XPD/Rad- 3样解旋酶（FANCJ、XPD、RTEL 1和CHLR 1）有助于基因组稳定性，如果受损， 导致各种遗传疾病和癌症风险增加。holC基因编码chi，这是一种 属于两种不同的复合体。Chi是DNA聚合酶III夹装载器的辅助亚基， 与全酶的复合物。Chi还结合YoaA以产生功能性YoaA-chi解旋酶。众所周知， 结合单链DNA结合蛋白（SSB），这种相互作用是解决停滞病变所必需的 复制的SSB是在生命的所有领域中发现的必需蛋白质，包覆单链（ss）DNA，并且 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， 解决中断复制的损坏是未知的。因此，本奖学金旨在描述SSB 与YoaA-chi的相互作用，以了解这种新的修复途径。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叉显示，YoaA-chi的解旋酶活性在 SSB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， 通过YoaA上可能的新相互作用（目的1）。由于SSB调节多种DNA结合蛋白， 通过各种机制，几个方面的YoaA-chi，SSB可以调节将被调查。将 确定SSB是否改变YoaA-chi的底物亲和力（aim 2）或YoaA-chi的解旋酶活性（aim 3）。这将是第一次研究SSB如何调节YoaA-chi以及这些蛋白质在一个特定的环境中的作用。 DNA修复机制。这项研究也将为我的培训做出重大贡献， 一个独立的生物化学家和佛罗里达大学的环境将使我成功。