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.

项目总结/文摘