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 损伤的准确修复对于细胞的生存和维持其基因组的稳定性至关重要。双链 DNA 断裂 (DSB) 是最致命的 DNA 损伤，细胞已进化出多种虽然某些 DSB 修复途径是准确的，但其他途径可能会破坏 DSB 的稳定性。通过创建与癌症和其他人类相关的突变或染色体重排来改造基因组我们的长期目标是找出驱动 DSB 修复进入有害 DNA 漩涡的因素。我们重点关注两种此类高风险 DSB 修复。途径：1) 断裂诱导复制 (BIR)，一种不寻常的长链修复 DNA 合成类型，可促进遗传不稳定性的爆发；2) 微同源介导的 BIR (MMBIR)，一种涉及的复制途径在微同源性位置发生多个模板转换事件，产生复杂的基因组我们将使用一种特殊的、强大的酵母系统来研究位点的修复- 特定的 HO-核酸内切酶诱导的 DSB 为其他系统中的研究设计提供信息。使我们在 BIR 和 MMBIR 的表征方面取得了重大进展，包括开发了几个其中一个创新工具，我们将其命名为 AMBER（监测 BIR 伸长率的测定），是一种基于液滴数字 PCR 的方法使用 AMBER 以前所未有的分辨率测量 BIR 动力学。在下一个 MIRA 支持周期中，我们将能够确定由我们控制的 BIR 的具体步骤我们还将使用新发现的 BIR 驱动蛋白候选物，包括纺锤体组装检查点蛋白。我们敏感的酵母 BIR 系统中的 AMBER 可解开碰撞后 BIR 调节的机制具有各种复制障碍，包括表征 Rad52 依赖性单链的作用碰撞后 BIR 重新启动的退火所获得的结果将有助于阐明 Rad51- 的机制。酵母中独立的 BIR，该途径可能类似于哺乳动物中描述的 BIR 事件。我们在 MIRA 支持下开发的另一种方法可以根据长周期检测 BIR 事件 APOBEC（胞苷脱氨酶）存在时由 BIR 形成的突变簇，我们提出在此应用此方法来检测酵母减数分裂期间的 BIR 诱变频率。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出生缺陷。最后，我们的新软件 MMBSearch（根据我们对酵母中 MMBIR 的表征而开发）将用于识别使人类细胞易受 MMBIR 事件影响的特定条件，我们最近发现在癌症中很常见，但在非癌细胞中很少见，将 MMBSearch 应用于全基因组测序。数据将识别特定的癌症、细胞类型、染色体位置和环境压力因素，从而促进 MMBIR。总体而言，该研究计划将产生有关促进风险因素的基础知识。 DSB 修复途径以及这些途径的机制可能会破坏真核基因组的稳定性。

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.