Double strand break repair maelstrom: causes, mechanisms and genome destabilizing consequences

双链断裂修复漩涡：原因、机制和基因组不稳定后果

基本信息

批准号：
10623641
负责人：
Anna L Malkova
金额：
$ 44.09万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-06 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10623641
关键词：
Automobile Driving Biological Assay Cell Survival Cells Chromosomes Complex Computer software Congenital Abnormality Cytidine Deaminase DNA Double Strand Break DNA Repair DNA Repair Pathway DNA Sequence Rearrangement DNA biosynthesis DNA lesion Data Detection Development Double Strand Break Repair Event Genetic Genome Genome Stability Goals HO nuclease Human Kinetics Knowledge Malignant Neoplasms Mammals Measures Mediating Meiosis Methodology Methods Molecular Monitor Mutation Names Neurologic Pathway interactions Pattern Positioning Attribute Proteins RAD52 gene Regulation Research Resolution Role Site Syndrome System Work Yeasts cell type chromosomal location data mining design digital early detection biomarkers environmental stressor genome sequencing high risk human disease innovation programs repaired targeted treatment tool whole genome

项目摘要

Accurate repair of DNA lesions is paramount to the survival of cells and to maintain their genomic stability. Double-strand DNA breaks (DSBs) are the most lethal DNA lesion, and cells have evolved a variety of mechanisms for their repair. While some DSB repair pathways are accurate, others can destabilize the genome by creating mutations or chromosome rearrangements associated with cancer and other human diseases. Our long-term goal is to identify factors that drive DSB repair into the maelstrom of deleterious DNA repair pathways, and to characterize their molecular mechanisms. We focus on two such high-risk DSB repair pathways: 1) break-induced replication (BIR), an unusual type of long-tract repair DNA synthesis that promotes bursts of genetic instabilities; and 2) microhomology-mediated BIR (MMBIR), a replicative pathway involving multiple template-switching events at positions of microhomologies that yields complex genomic rearrangements. We will use an extensively characterized, powerful yeast system to study repair of a site- specific HO-endonuclease-induced DSB to inform the design of studies in other systems. MIRA support enabled significant progress in our characterization of BIR and MMBIR, including development of several innovative tools. One of them, which we named AMBER (Assay for Monitoring BIR Elongation Rate), is a droplet-digital-PCR-based method to measure BIR kinetics with unprecedented resolution. Using AMBER during the next MIRA support cycle will allow us to identify the specific steps of BIR that are controlled by our newly identified BIR driver protein candidates, including spindle assembly checkpoint proteins. We will also use AMBER in our sensitive yeast BIR system to unravel the mechanisms of BIR regulation following its collision with various replication obstacles, including characterizing the role of Rad52-dependent single-strand annealing for BIR re-start after collision. The obtained results will shed light on the mechanism of Rad51- independent BIR in yeast, which is a pathway that is likely similar to BIR events described in mammals. Another approach that we developed with MIRA support enabled the detection of BIR events based on long mutation clusters formed by BIR occurring in the presence of APOBEC (cytidine deaminase), and we propose to apply this methodology here to detect BIR during yeast meiosis. Determining how frequently mutagenic BIR might be used to repair meiotic DSBs is important because similar events can lead to birth defects in humans. Finally, our new software, MMBSearch—developed based on our characterization of MMBIR in yeast—will be used to identify specific conditions that predispose human cells to MMBIR events, which we recently found to be frequent in cancer, but rare in non-cancerous cells. Applying MMBSearch to whole-genome sequencing data will identify specific cancers, cell types, chromosomal locations and environmental stressors that promote MMBIR. Overall, this research program will produce fundamental knowledge on the factors that promote risky DSB repair pathways and the mechanisms of these pathways that can destabilize eukaryotic genomes.

DNA病变的准确修复对于细胞的存活和维持其基因组稳定性至关重要。双链DNA断裂（DSB）是最致命的DNA病变，细胞已经进化了多种维修的机制。虽然某些DSB维修途径是准确的，但其他DSB可以使通过创建与癌症和其他人有关的突变或染色体重排来进行基因组疾病。我们的长期目标是确定将DSB维修推向有害DNA漩涡的因素修复途径，并表征其分子机制。我们专注于两个这样的高风险DSB修复途径：1）突发诱导的复制（BIR），这是一种不寻常的长项修复DNA合成，可促进遗传不稳定爆发； 2）微学介导的BIR（MMBIR），涉及的复制途径在微观理学位置的多个模板切换事件，产生复杂的基因组重排。我们将使用广泛特征的功能强大的酵母系统来研究站点的维修特定的HO-核酸酶诱导的DSB，以告知其他系统中的研究设计。 mira支持在我们对BIR和MMBIR的表征方面取得了重大进展，包括开发几个创新工具。其中之一，我们将其命名为Amber（用于监视Bir伸长率的测定），是基于液滴 - 数字PCR的方法，以前所未有的分辨率测量BIR动力学。使用琥珀在下一个MIRA支持周期中，我们可以确定BIR的特定步骤新确定的BIR驱动蛋白候选物，包括主轴组件检查点蛋白。我们还将使用我们敏感的酵母BIR系统中的琥珀色，以揭示BIR碰撞后的机制具有各种复制障碍，包括表征依赖Rad52的单链的作用碰撞后退火重新开始。获得的结果将阐明Rad51-的机制酵母中的独立BIR，这是一种可能与哺乳动物中描述的BIR事件相似的途径。我们使用MIRA支持开发的另一种方法使得基于长期的BIR事件检测由BIR形成的突变簇在存在APOBEC（胞苷脱氨酶）的情况下发生，我们建议在此处应用这种方法以检测酵母减数分裂过程中的BIR。确定诱变BIR的频率可能用于修复减数分裂DSB很重要，因为类似的事件可能导致人类的先天缺陷。最后，我们的新软件MMBSEarch（根据我们对酵母中MMBIR的特征开发）将是用于确定使人类细胞易受MMBIR事件的特定条件，我们最近发现经常在癌症中，但在非癌细胞中很少见。将MMBSEarch应用于全基因组测序数据将确定促进的特定癌症，细胞类型，染色体位置和环境压力源 mmbir。总体而言，该研究计划将对促进风险的因素产生基本知识 DSB修复途径和这些途径可能破坏真核基因组的机制。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Quantitative assessment reveals the dominance of duplicated sequences in germline-derived extrachromosomal circular DNA.

DOI：
10.1073/pnas.2102842118
发表时间：
2021-11-23
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Mouakkad-Montoya L;Murata MM;Sulovari A;Suzuki R;Osia B;Malkova A;Katsumata M;Giuliano AE;Eichler EE;Tanaka H
通讯作者：
Tanaka H

Measuring the contributions of helicases to break-induced replication.

测量解旋酶对断裂诱导复制的贡献。

DOI：
10.1016/bs.mie.2022.02.025
发表时间：
2022
期刊：
Methods in enzymology
影响因子：
0
作者：
Yan,Zhenxin;Liu,Liping;Pham,Nhung;Thakre,PilendraK;Malkova,Anna;Ira,Grzegorz
通讯作者：
Ira,Grzegorz

Break-Induced Replication: The Where, The Why, and The How.