The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

IF 2.1 3区 生物学 Q3 GENETICS & HEREDITY G3: Genes|Genomes|Genetics Pub Date : 2024-10-14 DOI:10.1093/g3journal/jkae210
Makenzie S Thomas, Gautham S Pillai, Margaret A Butler, Joel Fernandez, Jeannine R LaRocque
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Abstract

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

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黑腹果蝇 CtIP 和 Rif1 在 DNA 双链断裂同源定向修复中的表观关系
双链断裂(DSB)是对基因组稳定性构成重大威胁的遗传毒性 DNA 病变,需要精确高效的修复机制来防止细胞死亡或突变。DSB通过非同源末端连接(NHEJ)或同源定向修复(HDR)进行修复,其中包括同源重组(HR)和单链退火(SSA)。CtIP 和 Rif1 是与 DSB 修复途径选择有关的保守蛋白,可能是通过在促进 HDR 所需的 DNA 末端切除中发挥冗余作用。虽然这些蛋白的作用已在其他生物体中得到充分证实,但 Rif1 的作用及其与 CtIP 的潜在冗余在黑腹果蝇中的作用仍未确定。为了研究DmCtIP和DmRif1在DSB修复中的作用,本研究采用了白色直接重复(DR-white)检测法、通过分解(TIDE)分析法和P{wIw_2 kb 3'}检测法来分别跟踪HR、NHEJ和SSA的修复结果。这些实验是在 DmCtIPΔ/Δ 单突变体、DmRif1Δ/Δ 单突变体和 DmRif1Δ/Δ; DmCtIPΔ/Δ 双突变体中进行的。这项工作表明,DmCtIPΔ/Δ和DmRif1Δ/Δ单突变体在HR和SSA修复方面都存在明显缺陷。然而,DmRif1Δ/Δ;DmCtIPΔ/Δ双突变体的实验表明,DmCtIP在促进HDR方面与DmRif1具有外显性。总之,这项研究得出的结论是,DmRif1 和 DmCtIP 并非在一个冗余途径中执行其活动,而是 DmCtIP 是促进 HR 和 SSA 的主要驱动力,很可能是通过其在末端切除中的作用。
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来源期刊
G3: Genes|Genomes|Genetics
G3: Genes|Genomes|Genetics GENETICS & HEREDITY-
CiteScore
5.10
自引率
3.80%
发文量
305
审稿时长
3-8 weeks
期刊介绍: G3: Genes, Genomes, Genetics provides a forum for the publication of high‐quality foundational research, particularly research that generates useful genetic and genomic information such as genome maps, single gene studies, genome‐wide association and QTL studies, as well as genome reports, mutant screens, and advances in methods and technology. The Editorial Board of G3 believes that rapid dissemination of these data is the necessary foundation for analysis that leads to mechanistic insights. G3, published by the Genetics Society of America, meets the critical and growing need of the genetics community for rapid review and publication of important results in all areas of genetics. G3 offers the opportunity to publish the puzzling finding or to present unpublished results that may not have been submitted for review and publication due to a perceived lack of a potential high-impact finding. G3 has earned the DOAJ Seal, which is a mark of certification for open access journals, awarded by DOAJ to journals that achieve a high level of openness, adhere to Best Practice and high publishing standards.
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