IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2026-02-01 DOI: 10.1016/j.dnarep.2026.103923

Louise Juhl , Linea Busch , Jonas Bagge , Ruiqi Xu , Milorad Kojic , Mira Milisavljevic , Vibe H. Oestergaard , William K. Holloman , Michael Lisby

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSB), recovery of broken replication forks and formation of meiotic crossovers. HR provides a mechanism to precisely repair damaged DNA in a template-dependent process. The defining step in HR is homologous strand exchange directed by the RecA-related recombinase Rad51. BRCA2 and Brh2, the BRCA2 orthologue in Ustilago maydis, enable recombinational repair of DNA by controlling Rad51. In turn, Dss1, a small intrinsically disordered protein that binds to the C-terminal region of BRCA2/Brh2, regulates BRCA2/Brh2. In the present study, we dissect the interdependency of HR proteins for recruitment to DNA-damage induced foci using fluorescence microscopy and genetics. In U. maydis, Brh2 and Dss1 colocalize at DNA damage-induced foci. Dss1 recruitment to foci is dependent on interaction with full-length Brh2 and Dss1-Brh2 interaction is required for resistance to DNA damage. Further, Dss1 is required for Rad51 and Rec2 focus formation. Interestingly, we find that Rad52 is required for Brh2, Rec2 and Dss1 focus formation. In avian DT40 cells, we likewise show that endogenously tagged DSS1 redistributes into subnuclear foci in response to DNA damaging agents. However, DSS1 foci rarely colocalize with BRCA2 foci. Finally, Dss1 focus formation is inhibited by treatment with the proteasome inhibitor MG132, in both U. maydis and DT40 cells, suggesting a role of ubiquitin in homology-dependent repair.

同源重组（HR）是DNA双链断裂（DSB）修复、断裂复制叉恢复和减数分裂交叉形成的主要途径。HR提供了一种在模板依赖过程中精确修复受损DNA的机制。HR的决定性步骤是由reca相关重组酶Rad51引导的同源链交换。黑穗病菌BRCA2同源基因BRCA2和Brh2通过控制Rad51实现DNA的重组修复。反过来，Dss1，一种结合BRCA2/Brh2 c端区域的小的内在无序蛋白，调节BRCA2/Brh2。在本研究中，我们使用荧光显微镜和遗传学分析了HR蛋白在dna损伤诱导的病灶招募中的相互依赖性。在美国，Brh2和Dss1在DNA损伤诱导的病灶上共定位。Dss1向病灶的募集依赖于与全长Brh2的相互作用，Dss1-Brh2相互作用是抵抗DNA损伤所必需的。此外，Dss1是Rad51和Rec2焦点形成所必需的。有趣的是，我们发现Rad52是Brh2， Rec2和Dss1焦点形成所必需的。在禽类DT40细胞中，我们同样发现内源性标记的DSS1在DNA损伤剂的作用下重新分布到亚核病灶。然而，DSS1位点很少与BRCA2位点共定位。最后，在U. maydis和DT40细胞中，蛋白酶体抑制剂MG132可以抑制Dss1病灶的形成，这表明泛素在同源依赖性修复中的作用。

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引用次数: 0

Search, verify and excise-lesion recognition in NER 在NER中搜索、验证和切除病变识别

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2026-01-12 DOI: 10.1016/j.dnarep.2026.103922

Jochen Kuper, Caroline Kisker

Nucleotide excison repair (NER) is a highly versatile template-based DNA repair mechanism that can address lesions ranging from cyclo butane pyrimdine dimers, to cisplatinum crosslinks, and bulky adducts like acetyl amino flourenes. Eukaryotic NER employs more than thirty different proteins that coordinate and facilitate the search, verification, incision, and DNA re-synthesis of the various lesions with extremely high precision. High resolution structures of key complexes combined with biochemistry and computational biology have greatly enhanced our understanding of the NER process. In this review we will highlight recent discoveries in NER research concerning lesion search and handover of complex assemblies, lesion verification, and potential mechanisms providing the signal for incision with a focus on structural biology.

核苷酸切除修复（NER）是一种高度通用的基于模板的DNA修复机制，可以解决从环丁烷嘧啶二聚体到顺铂交联和大体积加合物如乙酰基氨基芴的损伤。真核生物NER使用超过30种不同的蛋白质，以极高的精度协调和促进各种病变的搜索、验证、切割和DNA重新合成。关键配合物的高分辨率结构与生物化学和计算生物学相结合，极大地增强了我们对NER过程的理解。在这篇综述中，我们将重点介绍最近在NER研究中的发现，包括病变搜索和复杂组件的移交、病变验证以及为切口提供信号的潜在机制，重点是结构生物学。

引用次数: 0

Genetic analysis reveals a timing-dependent functional interplay between Polζ and Polη in translesion DNA synthesis upon UV damage 遗传分析揭示了在UV损伤下翻译DNA合成中Polζ和Polη之间的时间依赖功能相互作用。

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2026-01-01 DOI: 10.1016/j.dnarep.2025.103919

Mone Okuda, Minori Fujii, Ryotaro Kawasumi, Kouji Hirota

Translesion DNA synthesis (TLS) plays a crucial role in restarting stalled replication at damaged templates. This process is facilitated by specialized DNA polymerases, such as Polη and Polζ, where Polη inserts nucleotides opposite the damaged template, and Polζ extends the primer following insertion. TLS occurring at the stalled replication fork is termed "on-the-fly" TLS, whereas TLS that fills gaps remaining after fork progression is referred to as "post-replicative gap-filling" TLS. However, the roles of Polη and Polζ in these two phases of TLS remain unclear. Here, we demonstrate the functional relationship between Polη and Polζ in these TLS pathways through genetic studies in human cells. We established POLH^−/−, REV3L^−/− (deficient in Polζ catalytic subunit, Rev3), and POLH^−/−/REV3L^−/− cells from human TK6 cells and evaluated the sensitivity of these cell lines to ultraviolet (UV). We found that the loss of Polη in REV3L^−/− cells led to the synergistic increase of the UV sensitivity, accompanied by a marked rise in UV-induced chromosomal aberrations. However, such synergistic effects were not observed in the rate of replication fork stalling after UV damage in POLH^−/−/REV3L^−/− cells. In marked contrast, the number of unrepaired gaps following UV irradiation was significantly increased in the double mutant. These findings suggest that Polη and Polζ function complementarily in promoting post-replicative gap-filling TLS while they act collaboratively in on-the-fly TLS. Our current genetic study in human cells revealed a previously unappreciated functional relationship between Polη and Polζ and showed the pivotal role of "post-replicative gap-filling" TLS in UV-tolerance.

翻译DNA合成（TLS）在重新启动受损模板的停滞复制中起着至关重要的作用。这一过程是由特殊的DNA聚合酶促进的，如Polη和Polζ，其中Polη插入与受损模板相反的核苷酸，Polζ在插入后延伸引物。发生在停止复制分叉处的TLS被称为“即时”TLS，而在分叉进展后填补剩余空白的TLS被称为“复制后填补空白”TLS。然而，Polη和Polζ在这两相TLS中的作用尚不清楚。在这里，我们通过人类细胞的遗传研究证明了Polη和Polζ在这些TLS通路中的功能关系。我们从人TK6细胞中建立了POLH-/-， REV3L-/-（缺乏Polζ催化亚基，Rev3）和POLH-/-/REV3L-/-细胞，并评估了这些细胞系对紫外线（UV）的敏感性。我们发现，在REV3L-/-细胞中Polη的缺失导致紫外线敏感性的协同增加，并伴随着紫外线诱导的染色体畸变的显著增加。然而，在紫外线损伤后POLH-/-/REV3L-/-细胞的复制叉停滞率中没有观察到这种协同效应。与此形成鲜明对比的是，双突变体在紫外线照射后未修复的间隙数量显著增加。这些发现表明，Polη和Polζ在促进复制后补隙的TLS中是互补的，而在动态TLS中是协同作用的。我们目前在人类细胞中的遗传学研究揭示了Polη和Polζ之间以前未被认识到的功能关系，并显示了“复制后间隙填充”TLS在紫外线耐受性中的关键作用。

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引用次数: 0

Cutting edge perspectives in genome maintenance XII 基因组维持的前沿观点。

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2026-01-01 DOI: 10.1016/j.dnarep.2025.103920

Penny Jeggo

引用次数: 0

Efficient activity of uracil DNA glycosylase (UNG2) in proliferating cells requires binding to proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) 尿嘧啶DNA糖基化酶（UNG2）在增殖细胞中的有效活性需要与增殖细胞核抗原（PCNA）和复制蛋白A （RPA）结合。

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2026-01-01 DOI: 10.1016/j.dnarep.2025.103918

Rashmi S. Kulkarni , Brian P. Weiser

The compounds pemetrexed and 5-fluorodeoxyuridine (FdU) are widely used for cancer therapies and disrupt cell proliferation by inducing DNA damage and stressing DNA replication. The drugs disrupt pyrimidine nucleotide metabolism and promote the accumulation of uracil bases in genomic DNA, which are repaired by uracil DNA glycosylase (UNG2) and downstream base excision repair proteins. UNG2 interacts with Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which localize to the replication fork during DNA damage responses to orchestrate DNA repair. In this work, we tested whether UNG2 requires interaction with PCNA and RPA to repair DNA damage in a colorectal cancer model during treatment with pemetrexed or FdU. We genetically knocked out UNG2 in HT29 cells and engineered the cells to express UNG2 variants that cannot bind to PCNA or RPA. We found that eliminating UNG2 activity or disrupting its interaction with PCNA or RPA sensitized the cells to the DNA-damaging effects of pemetrexed and FdU. The ability of UNG2 to localize to stalled replication forks was impaired when the enzyme could not interact with PCNA or RPA. Finally, disrupting the interaction of UNG2 with PCNA or RPA sensitized the cells to the cytotoxicity of the drugs. We concluded that certain cancers may be sensitized to pemetrexed and FdU by directly inhibiting the enzymatic activity of UNG2, by depleting UNG2 levels in the cell, or by impairing UNG2 function by inhibiting its protein-protein interactions.

化合物培美曲塞和5-氟脱氧尿苷（FdU）广泛用于癌症治疗，并通过诱导DNA损伤和胁迫DNA复制来破坏细胞增殖。这些药物破坏嘧啶核苷酸代谢，促进尿嘧啶碱基在基因组DNA中的积累，尿嘧啶DNA糖基化酶（UNG2）和下游碱基切除修复蛋白修复尿嘧啶碱基。UNG2与增殖细胞核抗原（PCNA）和复制蛋白A （RPA）相互作用，在DNA损伤反应中定位于复制叉以协调DNA修复。在这项工作中，我们测试了在培美曲塞或FdU治疗期间，UNG2是否需要与PCNA和RPA相互作用来修复结直肠癌模型中的DNA损伤。我们通过基因敲除HT29细胞中的UNG2，并对细胞进行改造，使其表达不能与PCNA或RPA结合的UNG2变体。我们发现，消除UNG2活性或破坏其与PCNA或RPA的相互作用使细胞对培美曲塞和FdU的dna损伤作用敏感。当酶不能与PCNA或RPA相互作用时，UNG2定位到停滞复制叉的能力受损。最后，破坏UNG2与PCNA或RPA的相互作用使细胞对药物的细胞毒性敏感。我们得出结论，某些癌症可能通过直接抑制UNG2的酶活性，通过消耗细胞中的UNG2水平，或通过抑制其蛋白质-蛋白质相互作用而损害UNG2的功能，从而对培美曲塞和FdU致敏。

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引用次数: 0

Contents of previous 3 special issues in this series of perspectives 本系列专题前3期的内容

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2026-01-01 DOI: 10.1016/j.dnarep.2025.103921

Penny Jeggo

引用次数: 0

A tale of two mechanisms: Clarification of the pathway for MBD4 catalyzed glycosidic bond cleavage using MD and QM/MM calculations 通过MD和QM/MM计算，阐明了MBD4催化糖苷键裂解的途径

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-12-19 DOI: 10.1016/j.dnarep.2025.103917

Dylan J. Nikkel, Stacey D. Wetmore

DNA methylation to yield 5-methylcytosine (5mC) in CpG motifs plays a vital role in epigenetic regulation. However, deamination of 5mC results in canonical thymine (T) that requires methyl-CpG-binding domain protein 4 (MBD4) for repair. This important function has resulted in MBD4 being implicated in various human health disorders including MBD4-associated neoplasia syndrome and cancer resistance to 5-fluorouracil treatment. Nevertheless, the catalytic mechanism of MBD4 is poorly understood, with conflicting experimental observations resulting in multiple proposals. To provide atomic level structural details of the active site conformation and clarify the mechanistic pathway, this study uses a combination of adaptively biased molecular dynamics (abMD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations to map the MBD4 catalytic mechanism. Although our data indicate that the catalytic D560 residue is flexible in the active site, only one conformation facilitates 5mC excision. Despite some literature proposing the formation of a DNA−protein crosslinked intermediate, our modeling suggests catalysis is only viable through a deglycosylation mechanism that involves a water nucleophile attacking C1′ of T, with D560 activating the nucleophile and stabilizing the transition state and nucleobase departure facilitated by a network of hydrogen bonds. This proposal is fully consistent with experimental crystallographic, mutagenic, stereoscopic, and kinetic data, and aligns the MBD4 catalytic pathway with that characterized for several other monofunctional DNA glycosylases. By furthering our knowledge of MBD4 catalysis, this work will aid in the future development of treatments for MBD4-related genetic disorders and the rational design of transition state mimic inhibitors to enhance existing cancer therapies.

CpG基序中DNA甲基化产生5-甲基胞嘧啶（5mC）在表观遗传调控中起着至关重要的作用。然而，5mC的脱胺导致典型胸腺嘧啶(T)的产生，这需要甲基- cpg结合结构域蛋白4 （MBD4）进行修复。这一重要功能导致MBD4与多种人类健康疾病有关，包括MBD4相关的瘤变综合征和对5-氟尿嘧啶治疗的癌症耐药性。然而，人们对MBD4的催化机制知之甚少，实验观察结果相互矛盾，导致多种建议。为了提供活性位点构象的原子水平结构细节和阐明机理途径，本研究采用自适应偏态分子动力学（abMD）模拟和量子力学/分子力学（QM/MM）计算相结合的方法来绘制MBD4的催化机理。虽然我们的数据表明催化D560残基在活性位点是灵活的，但只有一种构象有利于5mC的切除。尽管一些文献提出了DNA -蛋白质交联中间体的形成，但我们的模型表明，催化作用只能通过一种脱糖基化机制来实现，该机制涉及一种水亲核试剂攻击T的C1 '， D560激活亲核试剂并稳定过渡态和氢键网络促进的核碱基离开。这一建议与实验晶体学、诱变、立体和动力学数据完全一致，并使MBD4的催化途径与其他几种单功能DNA糖基酶的催化途径一致。通过进一步了解MBD4的催化作用，这项工作将有助于MBD4相关遗传疾病的治疗方法的未来发展，以及过渡状态模拟抑制剂的合理设计，以增强现有的癌症治疗。

{"title":"A tale of two mechanisms: Clarification of the pathway for MBD4 catalyzed glycosidic bond cleavage using MD and QM/MM calculations","authors":"Dylan J. Nikkel, Stacey D. Wetmore","doi":"10.1016/j.dnarep.2025.103917","DOIUrl":"10.1016/j.dnarep.2025.103917","url":null,"abstract":"<div><div>DNA methylation to yield 5-methylcytosine (5mC) in CpG motifs plays a vital role in epigenetic regulation. However, deamination of 5mC results in canonical thymine (T) that requires methyl-CpG-binding domain protein 4 (MBD4) for repair. This important function has resulted in MBD4 being implicated in various human health disorders including MBD4-associated neoplasia syndrome and cancer resistance to 5-fluorouracil treatment. Nevertheless, the catalytic mechanism of MBD4 is poorly understood, with conflicting experimental observations resulting in multiple proposals. To provide atomic level structural details of the active site conformation and clarify the mechanistic pathway, this study uses a combination of adaptively biased molecular dynamics (abMD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations to map the MBD4 catalytic mechanism. Although our data indicate that the catalytic D560 residue is flexible in the active site, only one conformation facilitates 5mC excision. Despite some literature proposing the formation of a DNA−protein crosslinked intermediate, our modeling suggests catalysis is only viable through a deglycosylation mechanism that involves a water nucleophile attacking C1′ of T, with D560 activating the nucleophile and stabilizing the transition state and nucleobase departure facilitated by a network of hydrogen bonds. This proposal is fully consistent with experimental crystallographic, mutagenic, stereoscopic, and kinetic data, and aligns the MBD4 catalytic pathway with that characterized for several other monofunctional DNA glycosylases. By furthering our knowledge of MBD4 catalysis, this work will aid in the future development of treatments for MBD4-related genetic disorders and the rational design of transition state mimic inhibitors to enhance existing cancer therapies.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"157 ","pages":"Article 103917"},"PeriodicalIF":2.7,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145837925","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Polymerase theta: Genome protection through regulated deployment 聚合酶theta：通过调控部署的基因组保护

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-12-16 DOI: 10.1016/j.dnarep.2025.103916

Chelsea M. Smith , Gaorav P. Gupta

DNA Polymerase theta (Polθ, gene name POLQ) is the central enzyme of theta-mediated end-joining (TMEJ), an intrinsically mutagenic DNA double-strand break (DSB) repair pathway. Polθ is broadly conserved across most metazoan and plant species, suggesting an essential yet incompletely understood role in genome maintenance. POLQ-deficient organisms are viable but often accumulate genomic aberrations, such as micronuclei and fragile site expression. In cancer, however, Polθ activity is often dysregulated, driving chromosomal rearrangements characteristic of TMEJ hyperactivity. The finding that some cancers are dependent on hyperactive TMEJ has positioned Polθ as a therapeutic target, particularly in tumors with homologous recombination deficiency. The dual nature of Polθ—as both a genome-preserving and genome-destabilizing factor—reflects the importance of damage context and multi-layered regulatory mechanisms that ensure its precise deployment in normal cells, while the loss of these regulatory controls may be prevalent in cancer. This review synthesizes current knowledge on the DNA damage contexts that require Polθ for repair and how Polθ activity is restricted to these contexts to prevent catastrophic genome rearrangements while also not interfering with error-free DNA repair.

DNA聚合酶theta （Polθ，基因名POLQ）是theta介导的末端连接（TMEJ）的中心酶，TMEJ是一种内在诱变的DNA双链断裂（DSB）修复途径。Polθ在大多数后生动物和植物物种中广泛保守，表明它在基因组维持中起着重要但尚未完全了解的作用。缺乏polq的生物体是有活力的，但往往积累基因组畸变，如微核和脆弱位点表达。然而，在癌症中，Polθ活性经常失调，导致TMEJ过度活跃的染色体重排特征。一些癌症依赖于过度活跃的TMEJ，这一发现将Polθ定位为治疗靶点，特别是在同源重组缺陷的肿瘤中。polθ的双重性质——作为基因组保存因子和基因组不稳定因子——反映了损伤背景和多层调节机制的重要性，这些机制确保了它在正常细胞中的精确部署，而这些调节控制的丧失可能在癌症中普遍存在。这篇综述综合了目前关于需要Polθ进行修复的DNA损伤背景的知识，以及如何将Polθ的活性限制在这些背景中，以防止灾难性的基因组重排，同时又不干扰无错误的DNA修复。

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引用次数: 0

Base excision repair and homologous recombination are required for prevention of a chronic DNA damage response in Saccharomyces cerevisiae 碱基切除修复和同源重组是预防酿酒酵母慢性DNA损伤反应所必需的。

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-12-16 DOI: 10.1016/j.dnarep.2025.103915

Yogesh Nepal , Sanjida Ahmed , Armand M. Berry , Anika Mahmood , Cory L. Holland , L. Kevin Lewis

The chromosomes within eukaryotic cells experience many types of damage that are generated naturally via endogenous processes. The specific pathways that are most critical for repair of such endogenously produced DNA lesions have not been identified. Previous work revealed that budding yeast mutants deficient in double-strand break repair exhibit a persistent DNA damage checkpoint response leading to chronically high levels of G₂ phase cells, even in the absence of exogenous damaging agents. In the current study yeast mutants deficient in each of the five major DNA repair pathways were tested separately for the high G₂ cell phenotype. Cells with reduced homologous recombination (HR) and base excision repair (BER), but not nucleotide excision repair, mismatch repair or nonhomologous end-joining, displayed high levels of large-budded G₂ cells. BER mutants exhibiting this phenotype included apn1, apn2, ogg1, ung1, ntg1 and ntg2 cells. The persistent stress response was abolished by inactivation of the checkpoint gene RAD9. Cell cycling aberrations were increased synergistically in severely BER-deficient apn1 apn2 double mutants and strongly elevated in cells deficient in both HR and BER. apn1 apn2 rad52 cells were inviable but could be partially rescued by inactivation of UNG1. Transcription of the damage-inducible RNR3 (DIN1) gene was persistently activated in both BER and HR mutants. Like HR mutants, BER-deficient cells were larger in size and spent approximately three times longer in G₂ phase than wildtype cells. The results demonstrate that two pathways, HR and BER, are essential for repair of endogenously generated DNA lesions and prevention of a chronic cellular stress response.

真核细胞内的染色体经历了许多类型的损伤，这些损伤是通过内源性过程自然产生的。修复这种内源性产生的DNA损伤最关键的具体途径尚未确定。先前的研究表明，即使在没有外源性损伤剂的情况下，缺乏双链断裂修复的出芽酵母突变体也会表现出持续的DNA损伤检查点反应，导致长期高水平的G2期细胞。在目前的研究中，酵母突变体缺乏五种主要的DNA修复途径，分别测试了高G2细胞表型。同源重组（HR）和碱基切除修复（BER）减少的细胞，而非核苷酸切除修复、错配修复或非同源末端连接的细胞，显示出高水平的大芽G2细胞。表现出这种表型的BER突变包括apn1、apn2、ogg1、ung1、ntg1和ntg2细胞。检查点基因RAD9的失活消除了持续的应激反应。在严重BER缺陷的apn1 apn2双突变体中，细胞周期畸变协同增加，在HR和BER缺陷的细胞中，细胞周期畸变强烈增加。apn1, apn2， rad52细胞不能存活，但UNG1的失活可以部分挽救细胞。损伤诱导型RNR3 （DIN1）基因的转录在BER和HR突变体中持续激活。与HR突变体一样，ber缺陷细胞的体积更大，G2期的时间大约是野生型细胞的三倍。结果表明，两种途径，HR和BER，是修复内源性DNA损伤和预防慢性细胞应激反应所必需的。

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引用次数: 0

Stepwise DNA damage and repair mechanisms at replication forks in response to topoisomerase I inhibition 拓扑异构酶I抑制对复制叉的逐步DNA损伤和修复机制的影响

IF 2.7 3区生物学 Q2 GENETICS & HEREDITY

DNA Repair

Pub Date : 2025-12-11 DOI: 10.1016/j.dnarep.2025.103914

Sofie Østergård Bæk , Kristina Keuper , Giacomo Milletti , Alba Adelantado-Rubio , Michael Lisby , Jiri Bartek , Christoffel Dinant , Apolinar Maya-Mendoza

Camptothecin (CPT) and its derivative irinotecan inhibit DNA topoisomerase I (TOP1), inducing replication stress by stabilizing the TOP1 cleavage complex. This prevents DNA re-ligation, resulting in single-stranded breaks that, if unresolved, can cause DNA replication fork collapse and double-stranded breaks. Cells respond to TOP1 inhibitors through homologous recombination (HR) repair and fork protection, with RAD51 playing a central role. However, the full mechanisms of how cells react to TOP1 inhibitors are not fully understood. Here, we systematically investigated cellular responses to TOP1 inhibitors, assessing the effects on DNA damage repair (DDR), replication, and cell viability. Using state-of-the-art quantitative image-based cytometry and single-molecule analyses, we reveal a dose and time-dependent mechanistic switch in DDR pathways, which differentially affects DNA replication. While the replication forks arrest after minutes in the presence of CPT, unexpectedly, after two hours of CPT exposure, the fork speed is faster than in the controls. Furthermore, we explain some of the contrasting effects of the replication fork dynamics and DDR activation triggered by TOP1 inhibition. Finally, we identify cancer genetic vulnerabilities, such as HR deficiency, that may be exploitable with low-dose TOP1 inhibitors.

喜树碱（CPT）及其衍生物伊立替康抑制DNA拓扑异构酶I (TOP1)，通过稳定TOP1切割复合体诱导复制应激。这可以防止DNA重新连接，导致单链断裂，如果不解决，可能导致DNA复制叉崩溃和双链断裂。细胞通过同源重组（homologous recombination， HR）修复和分叉保护对TOP1抑制剂产生应答，RAD51起着核心作用。然而，细胞对TOP1抑制剂反应的完整机制尚不完全清楚。在这里，我们系统地研究了细胞对TOP1抑制剂的反应，评估了对DNA损伤修复（DDR）、复制和细胞活力的影响。利用最先进的定量图像细胞术和单分子分析，我们揭示了DDR通路中剂量和时间依赖的机制开关，这对DNA复制有不同的影响。虽然在CPT存在几分钟后复制分叉停止，但出乎意料的是，在CPT暴露两小时后，分叉速度比对照组快。此外，我们解释了一些由TOP1抑制触发的复制叉动力学和DDR激活的对比效应。最后，我们确定了低剂量TOP1抑制剂可能利用的癌症遗传脆弱性，如HR缺乏。

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引用次数: 0

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