DNA Repair最新文献_第6页

DNA polymerase beta expression in head & neck cancer modulates the poly(ADP-ribose)-mediated replication checkpoint DNA聚合酶β在头颈癌中的表达调节poly（adp -核糖）介导的复制检查点

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-06-01 DOI: 10.1016/j.dnarep.2025.103853

Md Maruf Khan , Wynand P. Roos , Charlotte R. Pearson , Stefan M. Leonard , Anusha Angajala , Denise Y. Gibbs , Jeffrey C. Liu , Camille Ragin , Robert W. Sobol

Head and Neck Squamous Cell Carcinoma (HNSCC) imposes a significant health burden, necessitating innovative therapeutic strategies to enhance treatment efficacy. Current treatments, such as surgery, radiation, and chemotherapy, have limited effectiveness and yield severe side effects, emphasizing the need for targeted therapies. We have focused on DNA polymerase beta (Polβ) and its roles in replication stress, cellular responses to DNA damaging-therapies, and DNA damage response modifiers. Our investigations reveal a regulatory role for base excision repair (BER) proteins, including Polβ, in the cellular response to inhibitors of poly(ADP-ribose) glycohydrolase (PARG), an enzyme involved in poly(ADP-ribose) (PAR) degradation. The inhibition of PARG, in HNSCC cells, elicits replication stress and activates the PAR-induced S-phase/ATR checkpoint, leading to a block to replication, cell cycle arrest, and the onset of apoptosis. However, Polβ overexpression mitigates this response, reducing replication-stress-induced PAR foci formation, suggesting a modulation of replication checkpoint activation. We found that PARG inhibitor treatment is ineffective on HNSCC cells that overexpress Polβ, implying that the PARG inhibitor-induced PAR and apoptotic response is dependent on the level of Polβ. Further, our in vitro experiments demonstrate that combining PARG and ATR/CHK1 inhibitors overcomes Polβ-mediated treatment resistance in HNSCC cells, producing synergistic effects compared to the individual treatment conditions. Our findings suggest a possible treatment paradigm for HNSCC, employing ATR or CHK1 inhibitors in combination with PARG inhibitors. This strategy offers a promising path for more effective HNSCC treatments, potentially overcoming Polβ-related resistance.

头颈部鳞状细胞癌（HNSCC）造成了巨大的健康负担，需要创新的治疗策略来提高治疗效果。目前的治疗方法，如手术、放疗和化疗，效果有限，并产生严重的副作用，强调需要靶向治疗。我们专注于DNA聚合酶β (Polβ)及其在复制应激、细胞对DNA损伤治疗的反应和DNA损伤反应修饰剂中的作用。我们的研究揭示了碱基切除修复（BER）蛋白，包括Polβ，在细胞对聚（adp -核糖）糖水解酶（PARG）抑制剂的反应中起调节作用，PARG是一种参与聚（adp -核糖）（PAR）降解的酶。在HNSCC细胞中，PARG的抑制引发复制应激并激活par诱导的s期/ATR检查点，导致复制受阻、细胞周期阻滞和细胞凋亡的发生。然而，Polβ过表达减轻了这种反应，减少了复制应激诱导的PAR焦点形成，表明复制检查点激活的调节。我们发现，PARG抑制剂对过表达Polβ的HNSCC细胞无效，这意味着PARG抑制剂诱导的PAR和凋亡反应依赖于Polβ的水平。此外，我们的体外实验表明，联合使用PARG和ATR/CHK1抑制剂可以克服pol β介导的HNSCC细胞耐药，与单独的治疗条件相比，可以产生协同效应。我们的研究结果提示了一种可能的HNSCC治疗模式，即使用ATR或CHK1抑制剂与PARG抑制剂联合使用。这一策略为更有效的HNSCC治疗提供了一条有希望的途径，有可能克服pol β相关的耐药性。

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引用次数: 0

Dynamics of chromatin factors RSF1, CENPS and CENPX at DNA damage sites 染色质因子RSF1、CENPS和CENPX在DNA损伤位点的动态变化

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-06-01 DOI: 10.1016/j.dnarep.2025.103850

Pritishkumar Tidke , Andrew Flaus , Helen Dodson

Chromatin has a major influence on the DNA damage response (DDR). Several chromatin-related factors participate in specialised DNA packaging during the DDR including the CENPS and CENPX histone fold proteins, also known as MHF1/2, and the chromatin remodelling factor RSF1 although their contribution has remained unclear. We defined a timeline for RSF1, CENPS, and CENPX recruitment at DNA double strand breaks (DSBs) induced in live HeLa cells by microirradiation and calibrated this to published data to clarify the potential for their involvement in the DDR. CENPS, CENPX and RSF1 are recruited with a half time of ∼100 s and removed with a half time of ∼2000 s. Enrichment for cell cycle phase revealed that this recruitment occurs in G1, S and G2 phases, but that its half time in G2 appears to be delayed and stronger than in G1. Integration of these observations with timelines for other DDR factors reveals that CENPS and CENPX recruitment occurs simultaneously immediately after ATM activation and RNF8-RNF168 activity. The removal of CENPS and CENPX is at a similar time to loading of RPA and assembly of RAD51. This places RSF1, CENPS and CENPX in the vicinity of DSBs at the time when nucleosomes are being actively remodelled during the chromatin-dependent early response to DNA damage involving pathway choice and resection, and their increased abundance at DSBs in G2 correlates with extended resection for HR.

染色质对DNA损伤反应（DDR）有重要影响。在DDR过程中，一些染色质相关因子参与了特殊的DNA包装，包括CENPS和CENPX组蛋白折叠蛋白，也称为MHF1/2，以及染色质重塑因子RSF1，尽管它们的作用尚不清楚。我们定义了RSF1、CENPS和CENPX在微辐射诱导的活HeLa细胞DNA双链断裂（DSBs）中募集的时间线，并将其与已发表的数据进行校准，以阐明它们参与DDR的可能性。CENPS、CENPX和RSF1的招募时间为~ 100 s，去除时间为~ 2000 s。细胞周期期的富集表明，这种募集发生在G1、S和G2期，但其在G2期的一半时间似乎比G1期延迟且更强。将这些观察结果与其他DDR因素的时间轴相结合，发现在ATM激活和RNF8-RNF168活动之后，CENPS和CENPX的招募同时发生。CENPS和CENPX的去除与RPA的加载和RAD51的组装同时进行。这将RSF1、CENPS和CENPX置于dsb附近，此时核小体在染色质依赖的DNA损伤早期反应中被积极重塑，涉及途径选择和切除，并且它们在G2中dsb的丰度增加与HR的延长切除相关。

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引用次数: 0

Development and characterization of a novel NEIL1 nanobody 一种新型NEIL1纳米体的研制与表征

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-22 DOI: 10.1016/j.dnarep.2025.103849

Marlo K. Thompson , Mark H. Eggers , Danielle Flores , Israel Valenzuela , Zhengrong Yang , Joel F. Andrews , Tom Johnsten , Aishwarya Prakash

Nei endonuclease VIII-like 1 (NEIL1) is a bifunctional human DNA glycosylase that catalyzes the first step of the base excision repair (BER) pathway by recognizing and excising oxidized bases, including thymine glycol and the further oxidation products of 7,8-dihydro-8-oxoguanine (8-oxoG), spiroiminodihydantoin, and guanidinohydantoin. Despite its critical role in maintaining genome stability, NEIL1 is expressed at relatively low endogenous cellular levels compared to other BER proteins such as OGG1, Polβ, and APE1. As a result, most cellular studies have relied on overexpression systems. Additionally, progress in studying NEIL1 has been hindered by the inconsistent availability and continuity of specific commercially available antibodies. To address this challenge, we developed single-domain nanobodies (VHHs) targeting NEIL1. A yeast 2 hybrid (Y2H) screen identified ten VHH hits with the top candidate, henceforth called A5, emerging multiple times. Here, we characterize the binding properties of A5 using a combination of biochemical and molecular techniques. Differential scanning fluorimetry and glycosylase activity assays indicate that recombinant A5 specifically stabilizes recombinantly expressed NEIL1, while not interfering with its glycosylase activity. Moreover, our data suggest that A5 preferentially binds to NEIL1’s N-terminal glycosylase domain rather than its C-terminal flexible tail, which is known to mediate protein-protein interactions. In live-cell imaging studies, an A5-mCherry chromobody colocalizes with NEIL1-GFP and is recruited to sites of laser-induced DNA damage, suggesting its potential as a molecular tool for visualizing NEIL1 dynamics. These findings establish A5 as a valuable probe for studying NEIL1 function and opens new avenues for exploring its role in DNA repair.

Nei核酸内切酶VIII-like 1 （NEIL1）是一种双功能的人类DNA糖基酶，通过识别和切除氧化碱基来催化碱基切除修复（BER）途径的第一步，包括胸腺嘧啶乙二醇和7,8-二氢-8-氧鸟嘌呤（8-oxoG）、spiroiminodihydantoin和guanidinohydantoin的进一步氧化产物。尽管NEIL1在维持基因组稳定性方面发挥着关键作用，但与OGG1、Polβ和APE1等其他BER蛋白相比，NEIL1的内源性细胞表达水平相对较低。因此，大多数细胞研究都依赖于过表达系统。此外，研究NEIL1的进展一直受到特定商业抗体不稳定的可用性和连续性的阻碍。为了解决这一挑战，我们开发了针对NEIL1的单域纳米体（vhs）。酵母2杂交（Y2H）筛选确定了10个VHH，其中最热门的候选基因（因此称为A5）多次出现。在这里，我们利用生物化学和分子技术的结合来表征A5的结合特性。差示扫描荧光法和糖基酶活性分析表明，重组A5特异性地稳定了重组表达的NEIL1，而不干扰其糖基酶活性。此外，我们的数据表明，A5优先结合NEIL1的n端糖基酶结构域，而不是其c端柔性尾，已知其介导蛋白-蛋白相互作用。在活细胞成像研究中，A5-mCherry染色体体与NEIL1- gfp共定位，并被招募到激光诱导的DNA损伤位点，这表明它有可能成为一种可视化NEIL1动力学的分子工具。这些发现确立了A5作为研究NEIL1功能的有价值的探针，并为探索其在DNA修复中的作用开辟了新的途径。

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引用次数: 0

Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage RNF146的WWE结构域的过表达可调节DNA损伤位点的多核苷酸动力学

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-21 DOI: 10.1016/j.dnarep.2025.103845

Rasha Q. Al-Rahahleh , Wynand P. Roos , Kate M. Saville , Joel F. Andrews , Zhijin Wu , Christopher A. Koczor , Aishwarya Prakash , Robert W. Sobol

Protein poly-ADP-ribosylation (PARylation) is a post-translational modification formed by transferring successive units of ADP-ribose to target proteins to form poly-ADP-ribose (PAR) chains. PAR plays a critical role in the DNA damage response (DDR) by acting as a signaling platform to promote the recruitment of DNA repair factors to the sites of DNA damage that bind via their PAR-binding domains (PBDs). Several classes of PBD families have been identified, which recognize distinct parts of the PAR chain. Proteins encoding PBDs play an essential role in conveying the PAR-mediated signal through their interaction with PAR chains, which mediates many cellular functions, including the DDR. The WWE domain, encoded in 12 human proteins, identifies the iso-ADP-ribose moiety of the PAR chain. PARylation is a heterogeneous structure that is highly dynamic in cells. Capturing the dynamics of PARylation is essential to understanding its role in the DDR, which can be achieved by expanding the tool kit for PAR detection and tracking mediated by the unique binding capability of various sensors. We recently described the WWE domain of RNF146 as a robust genetically encoded probe, when fused to EGFP, for the detection of PAR in live cells. Expanding on this, we used structural prediction tools to evaluate all of the WWE domains encoded in human proteins, evaluating each as molecular PAR probes in live cells. We demonstrate unique PAR dynamics when tracked by WWE-encoded PAR binding domains, in addition to an engineered macrodomain, that can be exploited for modulation of the PAR-dependent DNA damage response.

蛋白质多adp核糖基化（PARylation）是一种翻译后修饰，通过将连续的adp核糖单位转移到靶蛋白上，形成多adp核糖（PAR）链。PAR在DNA损伤反应（DDR）中起着至关重要的作用，它作为一个信号平台，促进DNA修复因子通过其PAR结合结构域（PBDs）聚集到DNA损伤位点。已经确定了几种PBD家族，它们识别PAR链的不同部分。编码pbd的蛋白质通过与PAR链的相互作用在传递PAR介导的信号中发挥重要作用，PAR链介导许多细胞功能，包括DDR。WWE结构域编码于12种人类蛋白质中，用于识别PAR链的isoadp -核糖部分。PARylation是细胞中高度动态的异质结构。捕获PARylation的动态对于理解其在DDR中的作用至关重要，这可以通过扩展各种传感器独特的结合能力介导的PAR检测和跟踪工具包来实现。我们最近将RNF146的WWE结构域描述为一种强大的遗传编码探针，当与EGFP融合时，用于检测活细胞中的PAR。在此基础上，我们使用结构预测工具来评估人类蛋白质中编码的所有WWE结构域，并将每个结构域作为活细胞中的分子PAR探针进行评估。我们展示了通过wwe编码的PAR结合域和工程大结构域跟踪的独特PAR动态，这些结构域可用于调节PAR依赖的DNA损伤反应。

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引用次数: 0

PARP1-PROTACs providing novel opportunities in precision oncology by targeting DNA damage response PARP1-PROTACs通过靶向DNA损伤反应为精确肿瘤学提供了新的机会

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-21 DOI: 10.1016/j.dnarep.2025.103851

Md Sadique Hussain, Gaurav Gupta

引用次数: 0

BRCA1 and BRCA2 in DNA damage and replication stress response: Insights into their functions, mechanisms, and implications for cancer treatment BRCA1和BRCA2在DNA损伤和复制应激反应中的作用、机制及其对癌症治疗的影响

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-13 DOI: 10.1016/j.dnarep.2025.103847

Ziqi Xu , Haihua Xie , Lizhi Song , Yuhua Huang , Jun Huang

Genomic stability is a cornerstone of cellular survival and proliferation. To counter the constant threat posed by endogenous and exogenous DNA-damaging agents, cells rely on a network of intricate mechanisms to safeguard DNA integrity and ensure accurate replication. Among these, the BRCA1 and BRCA2 tumor suppressor proteins play pivotal roles. While traditionally recognized for their involvement in homologous recombination repair and cell cycle checkpoints, emerging evidence highlights their essential functions in protecting stalled replication forks during replication stress. Mutations in BRCA1 or BRCA2 disrupt these critical functions, leading to compromised genome stability and an increased susceptibility to various cancers, particularly breast and ovarian cancers. This review provides a comprehensive analysis of the multifaceted roles of BRCA1 and BRCA2, focusing on their contributions to DNA damage responses and replication stress management. By elucidating the molecular pathways through which BRCA1 and BRCA2 operate, we aim to provide insights into their pivotal roles in maintaining genomic integrity and their implications for cancer treatment.

基因组稳定性是细胞存活和增殖的基石。为了对抗内源性和外源性DNA损伤剂带来的持续威胁，细胞依靠复杂的机制网络来保护DNA的完整性并确保准确的复制。其中，BRCA1和BRCA2肿瘤抑制蛋白起着举足轻重的作用。虽然传统上认为它们参与同源重组修复和细胞周期检查点，但新出现的证据强调了它们在复制应激期间保护停滞复制分叉的基本功能。BRCA1或BRCA2的突变破坏了这些关键功能，导致基因组稳定性受损，并增加了对各种癌症的易感性，特别是乳腺癌和卵巢癌。这篇综述全面分析了BRCA1和BRCA2的多方面作用，重点关注它们对DNA损伤反应和复制应激管理的贡献。通过阐明BRCA1和BRCA2运作的分子途径，我们旨在深入了解它们在维持基因组完整性方面的关键作用及其对癌症治疗的影响。

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引用次数: 0

Emerging connections: Poly(ADP-ribose), FET proteins and RNA in the regulation of DNA damage condensates 新出现的联系：聚（adp -核糖），FET蛋白和RNA在DNA损伤凝聚体的调节

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-08 DOI: 10.1016/j.dnarep.2025.103846

Silvia Lombardi, Mara Zilocchi, Roland Nicsanu, Silvia Maria Luisa Barabino

Our genome is exposed to thousands of DNA lesions every day, posing a significant threat to cellular viability. To deal with these lesions, cells have evolved sophisticated repair mechanisms collectively known as the DNA damage response. DNA double-strand breaks (DSBs) are very cytotoxic damages, and their repair requires the precise and coordinated recruitment of multiple repair factors to form nuclear foci. Recent research highlighted that these repair structures behave as biomolecular condensates, i.e. membraneless compartments with liquid-like properties. The formation of condensates is driven by weak, multivalent interactions among proteins and nucleic acids, and recent studies highlighted the roles of poly(ADP-ribose) (PAR) and RNA in regulating DSBs-related condensates. Additionally, the FET family of RNA-binding proteins (including FUS, EWS and TAF15), has emerged as a critical player in the DNA damage response, with recent evidence suggesting that FET proteins support the formation and dynamics of repair condensates. Notably, phase separation of FET proteins is implicated also in their pathological functions in cancer biology, highlighting the pervasive role of condensation. This review will provide an overview of biomolecular condensates at DSBs, focusing on the interplay among PAR and RNA in the spatiotemporal regulation of FET proteins at repair complexes. We will also discuss the role of FET condensates in cancer biology and how they are targeted for therapeutic purposes. The study of biomolecular condensates holds great promise for advancing our understanding of key cellular processes and developing novel therapeutic strategies, but requires careful consideration of potential challenges.

我们的基因组每天暴露在数千个DNA损伤中，对细胞生存能力构成重大威胁。为了处理这些损伤，细胞进化出了复杂的修复机制，统称为DNA损伤反应。DNA双链断裂（DSBs）是一种非常具有细胞毒性的损伤，其修复需要多种修复因子的精确和协调募集来形成核病灶。最近的研究强调，这些修复结构表现为生物分子凝聚体，即具有液体样特性的无膜隔室。凝析物的形成是由蛋白质和核酸之间的弱多价相互作用驱动的，最近的研究强调了聚adp核糖（PAR）和RNA在调节dsbs相关凝析物中的作用。此外，FET家族的rna结合蛋白（包括FUS、EWS和TAF15）在DNA损伤反应中发挥了关键作用，最近的证据表明，FET蛋白支持修复凝聚体的形成和动力学。值得注意的是，FET蛋白的相分离也涉及其在癌症生物学中的病理功能，突出了凝聚的普遍作用。本文将对dsb的生物分子凝聚体进行综述，重点介绍PAR和RNA在修复复合体中FET蛋白时空调控中的相互作用。我们还将讨论FET凝聚物在癌症生物学中的作用以及它们如何被靶向治疗。对生物分子凝聚物的研究对于促进我们对关键细胞过程的理解和开发新的治疗策略具有很大的希望，但需要仔细考虑潜在的挑战。

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引用次数: 0

Targeting base excision repair in precision oncology 精准肿瘤学中靶向碱基切除修复

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-01 DOI: 10.1016/j.dnarep.2025.103844

Nicola P. Montaldo , Hilde Loge Nilsen , Diana L. Bordin

Targeting the DNA damage response (DDR) is a key strategy in cancer therapy, leveraging tumour-specific weaknesses in DNA repair pathways to enhance treatment efficacy. Traditional treatments, such as chemotherapy and radiation, use a broad, damage-inducing approach, whereas precision oncology aims to tailor therapies to specific genetic mutations or vulnerabilities. The clinical success of PARP inhibitors has renewed the interest in targeting DNA repair as a therapeutic strategy. Expanding the precision oncology toolbox by targeting the base excision repair (BER) pathway presents a promising avenue for cancer therapy, particularly in tumours that rely heavily on this pathway due to deficiencies in other DNA repair mechanisms. This review discusses how targeting BER could improve treatment outcomes, particularly in DDR-defective cancers. With ongoing advancements in biomarker discovery and drug development, BER-targeted therapies hold significant potential for refining precision oncology approaches.

靶向DNA损伤反应（DDR）是癌症治疗的关键策略，利用肿瘤特异性DNA修复途径的弱点来提高治疗效果。传统的治疗方法，如化疗和放疗，使用广泛的、诱导损伤的方法，而精确肿瘤学的目标是针对特定的基因突变或脆弱性定制治疗方法。PARP抑制剂的临床成功重新燃起了靶向DNA修复作为治疗策略的兴趣。通过靶向碱基切除修复（BER）途径来扩展精确肿瘤学工具箱，为癌症治疗提供了一条有希望的途径，特别是在由于其他DNA修复机制缺乏而严重依赖该途径的肿瘤中。这篇综述讨论了靶向BER如何改善治疗结果，特别是在耐多药缺陷癌症中。随着生物标志物发现和药物开发的不断进步，ber靶向治疗在改进精确肿瘤学方法方面具有巨大的潜力。

引用次数: 0

Joining of DNA breaks- interplay between DNA ligases and poly (ADP-ribose) polymerases DNA断裂的连接- DNA连接酶和多聚核糖聚合酶之间的相互作用

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-01 DOI: 10.1016/j.dnarep.2025.103843

Nicolas Call, Alan E. Tomkinson

The joining of DNA single- and double-strand breaks (SSB and DSB) is essential for maintaining genome stability and integrity. While this is ultimately accomplished in human cells by the DNA ligases encoded by the LIG1, LIG3 and LIG4 genes, these enzymes are recruited to DNA breaks through specific interactions with proteins involved in break sensing and recognition and/or break processing. In this review, we focus on the interplay between the DNA break-activated poly (ADP-ribose) polymerases, PARP1 and PARP2, poly (ADP-ribose) (PAR) and the DNA ligases in DNA replication and repair. The most extensively studied example of this interplay is the recruitment of DNA ligase IIIα (LigIIIα) and other repair proteins to SSBs through an interaction between XRCC1, a scaffold protein and partner protein of nuclear LigIIIα, and PAR synthesized by PARP1 and to a lesser extent PARP2. Recently, these proteins have been implicated in a back-up pathway for joining Okazaki fragments that appears to have a critical function even in cells with no defect in the major LigI-dependent pathway. Finally, we discuss the effects of FDA-approved PARP1/2 inhibitors on DNA replication and repair in cancer and non-malignant cells and the potential utility of DNA ligase inhibitors as cancer therapeutics.

DNA单链和双链断裂（SSB和DSB）的连接对于维持基因组的稳定性和完整性至关重要。虽然这最终是通过由LIG1、LIG3和LIG4基因编码的DNA连接酶在人类细胞中完成的，但这些酶通过与断裂感知、识别和/或断裂加工中涉及的蛋白质的特定相互作用被招募到DNA断裂中。本文综述了DNA断裂激活聚（adp -核糖）聚合酶PARP1和PARP2、聚（adp -核糖）（PAR）与DNA连接酶在DNA复制和修复中的相互作用。这种相互作用的最广泛研究的例子是DNA连接酶IIIα (LigIIIα)和其他修复蛋白通过XRCC1（核LigIIIα的支架蛋白和伴侣蛋白）与PARP1合成的PAR （PARP2合成的PAR）相互作用募集到SSBs。最近，这些蛋白参与了连接Okazaki片段的备用途径，即使在主要依赖ligi的途径中没有缺陷的细胞中也具有关键功能。最后，我们讨论了fda批准的PARP1/2抑制剂对癌症和非恶性细胞中DNA复制和修复的影响，以及DNA连接酶抑制剂作为癌症治疗药物的潜在用途。

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引用次数: 0

Weaponizing CRISPR/Cas9 for selective elimination of cells with an aberrant genome 武器化CRISPR/Cas9选择性消除具有异常基因组的细胞

IF 3 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-05-01 DOI: 10.1016/j.dnarep.2025.103840

Sara Tavella , Alessia di Lillo , Anastasia Conti , Fabio Iannelli , Alexandra Mancheno-Ferris , Valentina Matti , Raffaella Di Micco , Fabrizio d’Adda di Fagagna

The CRISPR/Cas9 technology is a powerful and versatile tool to disrupt genes’ functions by introducing sequence-specific DNA double-strand breaks (DSBs). Here, we repurpose this technology to eradicate aberrant cells by specifically targeting silent and non-functional genomic sequences present only in target cells to be eliminated. Indeed, an intrinsic challenge of most current therapies against cancer and viral infections is the non-specific toxicity that they can induce in normal tissues because of their impact on important cellular mechanisms shared, to different extents, between unhealthy and healthy cells. The CRISPR/Cas9 technology has potential to overcome this limitation; however, so far effectiveness of these approaches was made dependent on the targeting and inactivation of a functional gene product. Here, we generate proof-of-principle evidence by engineering HeLa and RKO cells with a promoterless Green Fluorescent Protein (GFP) construct. The integration of this construct simulates either a genomic alteration, as in cancer cells, or a silent proviral genome. Cas9-mediated DSBs in the GFP sequence activate the DNA damage response (DDR), reduce cell viability and increase mortality. This is associated with increased cell size, multinucleation, cGAS-positive micronuclei accumulation and the activation of an inflammatory response. Pharmacological inhibition of the DNA repair factor DNA-PK enhances cell death. These results demonstrate the therapeutic potential of the CRISPR/Cas9 system in eliminating cells with an aberrant genome, regardless of the expression or the function of the target DNA sequence.

CRISPR/Cas9技术是一种强大而通用的工具，通过引入序列特异性DNA双链断裂（dsb）来破坏基因功能。在这里，我们重新利用这项技术，通过特异性靶向仅存在于要消除的靶细胞中的沉默和非功能基因组序列来根除异常细胞。事实上，目前大多数针对癌症和病毒感染的治疗方法的一个内在挑战是，它们可能在正常组织中诱导非特异性毒性，因为它们在不同程度上影响了不健康细胞和健康细胞之间共享的重要细胞机制。CRISPR/Cas9技术有潜力克服这一限制；然而，到目前为止，这些方法的有效性依赖于功能性基因产物的靶向和失活。在这里，我们通过用无启动子的绿色荧光蛋白（GFP）构建HeLa和RKO细胞来获得原理证明。这种结构的整合要么模拟了基因组的改变，如癌细胞，要么模拟了沉默的前病毒基因组。GFP序列中cas9介导的dsb激活DNA损伤反应（DDR），降低细胞活力，增加死亡率。这与细胞大小增加、多核、cgas阳性微核积累和炎症反应的激活有关。DNA修复因子DNA- pk的药理抑制促进细胞死亡。这些结果证明了CRISPR/Cas9系统在消除具有异常基因组的细胞方面的治疗潜力，而不管目标DNA序列的表达或功能如何。

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引用次数: 0