Molecular mechanisms of pathway choice in DNA double strand break repair

DNA双链断裂修复途径选择的分子机制

基本信息

批准号：
10646302
负责人：
Joseph J. Loparo
金额：
$ 38万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646302
关键词：
Address Back Binding Biochemical Biological Biological Assay Biological Process C-terminal Cell Death Cell Survival Cells Chromosomal Breaks Chromosomal Rearrangement Chromosomal translocation Chromosome Pairing DNA DNA Binding DNA Damage DNA Double Strand Break DNA Ligation DNA Repair Pathway Dangerousness Data Decision Making Double Strand Break Repair Enzymes Equilibrium Eukaryotic Cell Excision Filament Genes Glues Health Human Image Individual Invaded Knowledge Label Ligation Malignant Neoplasms Mastication Mediating Molecular N-terminal Nonhomologous DNA End Joining Nucleoproteins Nucleotides Pathway interactions Physiological Play Polymerase Process Proteins Rad51 recombinase Rana Reaction Regulation Resected Role Signal Transduction Single-Stranded DNA Sister Chromatid Site Synapses System Time Work Xenopus Xenopus laevis cancer cell cancer therapy clinically relevant cofactor egg experimental study genetic approach helicase homologous recombination imaging approach insertion/deletion mutation insight molecular imaging novel reconstitution repaired response single molecule targeted cancer therapy trait tumorigenesis

项目摘要

Project Summary DNA double strand breaks (DSBs) are a particularly toxic form of DNA damage. Even a single DSB can lead to cell death. Our cells use a number of intricate repair pathways to repair DSBs. For the majority of breaks that occur in our cells a pathway known as non-homologous end joining (NHEJ) is used to effectively “glue” the broken strands back together. Alternatively, a strand at each DNA end is “chewed back” in a process known as resection which generates single-stranded DNA overhangs. Multiple pathways act on these overhangs including homologous recombination (HR) and microhomology mediated end joining (MMEJ). HR is an intricate mechanism which uses a sister chromatid to direct repair in a high-fidelity manner while MMEJ is a mutagenic pathway whose mechanism more closely resembles NHEJ. Proper selection of these repair pathways is critical for human health as misuse is correlated with gross chromosomal changes that can result in cancer. Often cancer cells are deficient in DNA repair pathways which allows them to rapidly acquire traits not normally associated with healthy cells. These repair deficiencies also make them vulnerable to targeted cancer therapies. This proposal seeks to develop a better molecular understanding of how cells choose between these different DSB repair pathways. Experiments in cells have identified a number of proteins that play a role in this molecular decision-making process, but we lack an understanding of how these proteins work together. Traditionally, such knowledge is gained by purifying individual proteins and combining them together to reconstitute a biological process. However, given the sheer number of proteins, it is currently untenable to take such an approach. We have recently validated a cell-free extract made from the eggs of the frog Xenopus laevis as system that recapitulates DSB pathway choice. Combining this physiological biochemical system with powerful imaging approaches to study the dynamics of DNA DSB repair proteins at the single-molecule level, we will elucidate the molecular basis of DSB repair pathway choice. Specifically, we will work to clarify how the NHEJ factor Ku, which antagonizes DNA end resection, is removed from DNA ends (Aim 1); and how the MMEJ polymerase Pol θ competes with long-range resection on partially resected overhangs (Aim 2). Finally, we will elucidate how the multi-functional Pol θ uses its diverse enzymatic activities to synapse DNA ends and search for microhomology (Aim 3). Our studies will reveal significant new insights into DSB repair pathway selection and regulation. These insights may enable novel ways to alter the balance between these repair pathways which could have applications in gene editing or in therapies for cancers deficient in DSB repair.

项目概要 DNA 双链断裂 (DSB) 是一种毒性特别大的 DNA 损伤形式，即使是单个 DSB 也可能导致DNA 损伤。我们的细胞使用许多复杂的修复途径来修复 DSB。我们的细胞中发生了一种称为非同源末端连接（NHEJ）的途径，用于有效地“粘合” 或者，DNA 两端的一条链在称为“咀嚼”的过程中被“咀嚼回去”。产生单链 DNA 突出端的切除有多种途径作用于这些突出端。包括同源重组（HR）和微同源介导的末端连接（MMEJ）是一个复杂的过程。该机制使用姐妹染色单体以高保真方式直接修复，而 MMEJ 是一种诱变剂其机制与 NHEJ 更为相似，正确选择这些修复途径至关重要。对人类健康而言，因为滥用与可导致癌症的严重染色体变化相关。癌细胞缺乏 DNA 修复途径，这使得它们能够快速获得通常不具有的特征这些修复缺陷也使它们容易受到靶向癌症的影响。疗法。该提案旨在从分子角度更好地理解细胞如何在这些之间进行选择不同的 DSB 修复途径的细胞实验已经鉴定出许多在此过程中发挥作用的蛋白质。分子决策过程，但我们缺乏对这些蛋白质如何协同工作的了解。传统上，此类知识是通过纯化单个蛋白质并将它们组合在一起以形成然而，考虑到蛋白质的数量庞大，目前尚无法采取这种方法。我们最近验证了一种由爪蟾卵制成的无细胞提取物。 laevis 作为概括 DSB 途径选择的系统，将这种生理生化系统与该系统相结合。强大的成像方法可在单分子水平上研究 DNA DSB 修复蛋白的动态，我们将阐明 DSB 修复途径选择的分子基础，具体来说，我们将努力阐明如何选择。 NHEJ 因子 Ku 拮抗 DNA 末端切除，从 DNA 末端去除（目标 1）； MMEJ 聚合酶 Pol θ 与部分切除的突出部分的长距离切除竞争（目标 2）。我们将阐明多功能 Pol θ 如何利用其多种酶活性来突触 DNA 末端并寻找微同源性（目标 3）。我们的研究将揭示 DSB 修复途径的重要新见解。这些见解可能会带来改变这些修复之间平衡的新方法。这些途径可用于基因编辑或 DSB 修复缺陷的癌症治疗。