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; 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 从暴露于紫外光到有毒化学物质的外源性来源经常受到DNA的损害 环境。为了固定这些药物造成的损害并保持基因组稳定性，细胞具有多个 有效的DNA修复机制。但是，如果损坏负担，一些损坏将不可避免地逃脱维修 太高了。未修复的DNA损伤可以阻止DNA合成，并对细胞产生严重的后果 以及人类健康。 Brown等人的研究。使用叠氮噻噻替胺（AZT）作为阻止E.复制的工具。 大肠杆菌发现用于解决停滞的复制叉的必要基因。 AZT是胸苷类似物 在合成过程中掺入并防止底漆扩展，从而导致复制失速和单链 DNA间隙形成。发现两个基因YOAA和HOLC对于解决停滞的DNA复制至关重要 在AZT处理的大肠杆菌细胞中。 YOAA基因编码XPD/RAD3样解旋酶。四个人xpd/rad- 3像解旋酶（FANCJ，XPD，RTEL1和CHLR1）一样有助于基因组稳定性，如果受到损害，则可以 引起各种遗传疾病，并增加患癌症的风险。 HOLC基因编码CHI，这是一个 两个不同复合物的一部分。 CHI是DNA聚合酶III夹具加载器的附件亚基，并形成 与全酶的复合物。 CHI还结合YOAA以创建功能性的YoAA-CHI解旋酶。 Chi已知 结合单链DNA结合蛋白（SSB），这种相互作用对于解决停滞的病变是必需的 复制。 SSB是在生命的所有领域，涂层，单链（SS）DNA和 与十多种DNA修复和复制蛋白相互作用。 Yoaa，Chi和SSB如何合作 解决停止复制的损害尚不清楚。因此，该奖学金旨在表征SSB 与Yoaa-Chi与生化技术的相互作用，以了解这种新颖的修复途径。这是 假设的SSB调节Yoaa-Chi放松双链DNA解决病变的能力 基于初步数据的复制叉，该数据表明Yoaa-Chi的解旋酶活性在 SSB的存在。 SSB如何绑定Yoaa-Chi将被阐明，无论是通过CHI上的已知位置还是 通过可能在yoaa上进行的新互动（AIM 1）。因为SSB调节多种DNA结合蛋白 通过各种机制，将研究SSB可以调节的Yoaa-Chi的几个方面。这将是 确定SSB是否改变了YOAA-CHI（AIM 2）或YOAA-CHI的解旋酶活性的底物亲和力（AIM 3）。这将是SSB如何调节yoaa-chi的首次研究以及这些蛋白质在 新型的DNA修复机制。这项研究还将为我的培训做出重大贡献 佛罗里达大学的独立生物化学家和环境将使我成功。