Pub Date : 2024-10-26DOI: 10.1016/j.dnarep.2024.103780
Shuangyi Xu , Dieter Egli
A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.
正常发育需要一个基本稳定的基因组，尽管基因变化是繁殖、基因适应和进化不可或缺的一个方面。最近的研究突显了哺乳动物发育过程中的一个关键窗口期，即受精后第一次细胞分裂时，DNA 复制面临内在压力，基因组不稳定。哺乳动物的 DNA 复制模式和基因组稳定性很早就建立起来了，与核组织模式一起，在基因表达模式出现之前，在细胞规格化和生殖系形成之前。对哺乳动物胚胎中 DNA 复制和基因组稳定性的研究提供了一个独特的细胞系统，因为表观基因组重置为全能状态，核组织、基因表达、DNA 甲基化、组蛋白修饰和 DNA 复制模式从头建立。对哺乳动物早期胚胎的 DNA 复制和基因组稳定性进行研究，有助于了解正常和致病基因变异，并揭示基因组调控的基本原理。
{"title":"Genome organization and stability in mammalian pre-implantation development","authors":"Shuangyi Xu , Dieter Egli","doi":"10.1016/j.dnarep.2024.103780","DOIUrl":"10.1016/j.dnarep.2024.103780","url":null,"abstract":"<div><div>A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the <em>de novo</em> establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103780"},"PeriodicalIF":3.0,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142587374","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}
Pub Date : 2024-10-19DOI: 10.1016/j.dnarep.2024.103775
Nathan MacGilvary, Sharon B. Cantor
The mechanisms by which poly(ADP-ribose) polymerase 1 (PARP1) inhibitors (PARPi)s inflict replication stress and/or DNA damage are potentially numerous. PARPi toxicity could derive from loss of its catalytic activity and/or its physical trapping of PARP1 onto DNA that perturbs not only PARP1 function in DNA repair and DNA replication, but also obstructs compensating pathways. The combined disruption of PARP1 with either of the hereditary breast and ovarian cancer genes, BRCA1 or BRCA2 (BRCA), results in synthetic lethality. This has driven the development of PARP inhibitors as therapies for BRCA-mutant cancers. In this review, we focus on recent findings that highlight loss of PARP1 catalytic activity, rather than PARPi-induced allosteric trapping, as central to PARPi efficacy in BRCA deficient cells. However, we also review findings that PARP-trapping is an effective strategy in other genetic deficiencies. Together, we conclude that the mechanism-of-action of PARP inhibitors is not unilateral; with loss of activity or enhanced trapping differentially killing depending on the genetic context. Therefore, effectively targeting cancer cells requires an intricate understanding of their key underlying vulnerabilities.
聚（ADP-核糖）聚合酶 1（PARP1）抑制剂（PARPi）造成复制压力和/或 DNA 损伤的机制可能有很多。PARPi 的毒性可能来自其催化活性的丧失和/或 PARP1 在 DNA 上的物理诱捕，这不仅扰乱了 PARP1 在 DNA 修复和 DNA 复制中的功能，还阻碍了补偿途径。将 PARP1 与遗传性乳腺癌和卵巢癌基因 BRCA1 或 BRCA2（BRCA）中的任一基因结合破坏，会导致合成致死。这推动了 PARP 抑制剂作为 BRCA 突变癌症疗法的发展。在这篇综述中，我们将重点关注最近的研究结果，这些研究结果强调了 PARP1 催化活性的丧失，而不是 PARPi 诱导的异位捕获，是 PARPi 在 BRCA 基因缺失细胞中发挥疗效的核心原因。不过，我们也回顾了有关 PARP 诱捕在其他遗传缺陷中也是一种有效策略的研究结果。总之，我们得出的结论是，PARP 抑制剂的作用机制并不是单方面的；根据遗传背景的不同，活性丧失或诱捕增强的杀伤力也不同。因此，要有效地靶向癌细胞，就必须深入了解其关键的潜在弱点。
{"title":"Positioning loss of PARP1 activity as the central toxic event in BRCA-deficient cancer","authors":"Nathan MacGilvary, Sharon B. Cantor","doi":"10.1016/j.dnarep.2024.103775","DOIUrl":"10.1016/j.dnarep.2024.103775","url":null,"abstract":"<div><div>The mechanisms by which poly(ADP-ribose) polymerase 1 (PARP1) inhibitors (PARPi)s inflict replication stress and/or DNA damage are potentially numerous. PARPi toxicity could derive from loss of its catalytic activity and/or its physical trapping of PARP1 onto DNA that perturbs not only PARP1 function in DNA repair and DNA replication, but also obstructs compensating pathways. The combined disruption of PARP1 with either of the hereditary breast and ovarian cancer genes, <em>BRCA1</em> or <em>BRCA2</em> (BRCA), results in synthetic lethality. This has driven the development of PARP inhibitors as therapies for BRCA-mutant cancers. In this review, we focus on recent findings that highlight loss of PARP1 catalytic activity, rather than PARPi-induced allosteric trapping, as central to PARPi efficacy in BRCA deficient cells. However, we also review findings that PARP-trapping is an effective strategy in other genetic deficiencies. Together, we conclude that the mechanism-of-action of PARP inhibitors is not unilateral; with loss of activity or enhanced trapping differentially killing depending on the genetic context. Therefore, effectively targeting cancer cells requires an intricate understanding of their key underlying vulnerabilities.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103775"},"PeriodicalIF":3.0,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-19DOI: 10.1016/j.dnarep.2024.103777
Treshaun B. Sutton , Danielle L. Sawyer , Tasmin Naila , Joann B. Sweasy , Alan E. Tomkinson , Sarah Delaney
DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.
DNA 损伤是导致基因组不稳定的根本分子原因。碱基切除修复（BER）是将受损核碱基病变的潜在突变性和/或毒性降至最低的一道防线。然而，人们对染色质中的碱基切除修复尚未完全了解，在染色质中，真核生物基因组 DNA 是通过 DNA 组蛋白相互作用的层次结构压实的。在这里，我们研究了核糖体核心颗粒（NCP）中 27 个独特几何位置的 BER 酶的活性，核糖体核心颗粒是染色质中包装的最小单位。BER酶包括尿嘧啶DNA糖基化酶（UDG）、AP内切酶1（APE1）、DNA聚合酶β（Pol β）和与X射线修复交叉互补组1（LigIIIα/XRCC1）复合的DNA连接酶IIIα。这种对 BER 的全面分析表明，UDG 修复事件的启动是由病变的旋转位置决定的。APE1 在启动修复的位置具有强大的活性，而在核糖体 DNA 中央 ∼45 bp 的 Pol β 核苷酸掺入步骤中，修复活动停滞不前。修复的最后一步由 LigIIIα/XRCC1 催化，只有在 NCP 的入口/出口区域，当缺口位点从组蛋白上解开而瞬时暴露时才能完成。动力学测定进一步阐明，受损病变的位置会调节酶的活性。值得注意的是，这些数据表明，即使没有染色质重塑因子或其他细胞因子，一些 BER 酶也能在许多位置发挥作用。这些结果为 DNA 损伤和突变的全基因组图谱提供了信息，有助于我们了解突变热点和特征。
{"title":"Global screening of base excision repair in nucleosome core particles","authors":"Treshaun B. Sutton , Danielle L. Sawyer , Tasmin Naila , Joann B. Sweasy , Alan E. Tomkinson , Sarah Delaney","doi":"10.1016/j.dnarep.2024.103777","DOIUrl":"10.1016/j.dnarep.2024.103777","url":null,"abstract":"<div><div>DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103777"},"PeriodicalIF":3.0,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142549661","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}
Pub Date : 2024-10-19DOI: 10.1016/j.dnarep.2024.103778
Payel Dey, Rima Das, Sandipan Chatterjee, Roni Paul, Utpal Ghosh
The utilization of high linear energy transfer (LET) carbon ion (12C-ion) in radiotherapy has witnessed a notable rise in managing highly metastatic, recurrent, and chemo/radio-resistant human cancers. Non-small cell lung cancer (NSCLC) presents a formidable challenge due to its chemo-resistance and aggressive nature, resulting in poor prognosis and survival rates. In a previous study, we demonstrated that the combination of 12C-ion with the poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib significantly mitigated metastasis in A549 cells. Here, we delve into the underlying rationale behind the combined action of olaparib with 12C-ion, focusing on DNA repair pathways and cell death mechanisms in asynchronous NSCLC A549 cells following single and combined treatments. Evaluation included analysis of colony-forming ability, DNA damage assessed by γH2AX foci, expression profiling of key proteins involved in Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) repair pathways, caspase-3 activation, apoptotic body formation, and autophagic cell death. Our findings reveal that both PARPi olaparib and rucaparib sensitize A549 cells to 12C-ion exposure, with olaparib exhibiting superior sensitization. Moreover, 12C-ion exposure alone significantly downregulates both HR and NHEJ repair pathways by reducing the expression of MRE11--RAD51 and Ku70-Ku80 protein complexes at 24 h post-treatment. Notably, the combination of olaparib pre-treatment with 12C-ion markedly inhibits both HR and NHEJ pathways, culminating in DNA damage-induced apoptotic and autophagic cell death. Thus we are the first to demonstrate that olaparib sensitizes NSCLC cells to carbon ion by interfering with HR and NHEJ pathway. These insights underscore the promising therapeutic potential of combining PARP inhibition with carbon ion exposure for effective NSCLC management.
{"title":"Combined effects of carbon ion radiation and PARP inhibitor on non-small cell lung carcinoma cells: Insights into DNA repair pathways and cell death mechanisms","authors":"Payel Dey, Rima Das, Sandipan Chatterjee, Roni Paul, Utpal Ghosh","doi":"10.1016/j.dnarep.2024.103778","DOIUrl":"10.1016/j.dnarep.2024.103778","url":null,"abstract":"<div><div>The utilization of high linear energy transfer (LET) carbon ion (<sup>12</sup>C-ion) in radiotherapy has witnessed a notable rise in managing highly metastatic, recurrent, and chemo/radio-resistant human cancers. Non-small cell lung cancer (NSCLC) presents a formidable challenge due to its chemo-resistance and aggressive nature, resulting in poor prognosis and survival rates. In a previous study, we demonstrated that the combination of <sup>12</sup>C-ion with the poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib significantly mitigated metastasis in A549 cells. Here, we delve into the underlying rationale behind the combined action of olaparib with <sup>12</sup>C-ion, focusing on DNA repair pathways and cell death mechanisms in asynchronous NSCLC A549 cells following single and combined treatments. Evaluation included analysis of colony-forming ability, DNA damage assessed by γH2AX foci, expression profiling of key proteins involved in Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) repair pathways, caspase-3 activation, apoptotic body formation, and autophagic cell death. Our findings reveal that both PARPi olaparib and rucaparib sensitize A549 cells to <sup>12</sup>C-ion exposure, with olaparib exhibiting superior sensitization. Moreover, <sup>12</sup>C-ion exposure alone significantly downregulates both HR and NHEJ repair pathways by reducing the expression of MRE11--RAD51 and Ku70-Ku80 protein complexes at 24 h post-treatment. Notably, the combination of olaparib pre-treatment with <sup>12</sup>C-ion markedly inhibits both HR and NHEJ pathways, culminating in DNA damage-induced apoptotic and autophagic cell death. Thus we are the first to demonstrate that olaparib sensitizes NSCLC cells to carbon ion by interfering with HR and NHEJ pathway. These insights underscore the promising therapeutic potential of combining PARP inhibition with carbon ion exposure for effective NSCLC management.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103778"},"PeriodicalIF":3.0,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142561484","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}
Pub Date : 2024-10-19DOI: 10.1016/j.dnarep.2024.103776
Haichao Zhao , Christine Richardson , Ian Marriott , In Hong Yang , Shan Yan
To maintain genomic integrity, cells have evolved several conserved DNA damage response (DDR) pathways in response to DNA damage and stress conditions. Apurinic/apyrimidinic endonuclease 1 (APE1) exhibits AP endonuclease, 3′-5′ exonuclease, 3′-phosphodiesterase, and 3′-exoribonuclease activities and plays critical roles in the DNA repair and redox regulation of transcription. However, it remains unclear whether and how APE1 is involved in DDR pathways. In this perspective, we first updated our knowledge of APE1's functional domains and its nuclease activities and their specific associated substrates. We then summarized the newly discovered roles and mechanisms of action of APE1 in the global and nucleolar ATR-mediated DDR pathway. While the ATM-mediated DDR is well known to be activated by DNA double-strand breaks and oxidative stress, here we provided new perspectives as to how ATM DDR signaling is activated by indirect single-strand breaks (SSBs) resulting from genotoxic stress and defined SSB structures, and discuss how ATM kinase is directly activated and regulated by its activator, APE1. Together, accumulating body of new evidence supports the notion that APE1 is a master regulator protein of the ATR- and ATM-mediated DDR pathways. These new findings of APE1 in DDR signaling provide previously uncharacterized but critical functions and regulations of APE1 in genome integrity.
{"title":"APE1 is a master regulator of the ATR-/ATM-mediated DNA damage response","authors":"Haichao Zhao , Christine Richardson , Ian Marriott , In Hong Yang , Shan Yan","doi":"10.1016/j.dnarep.2024.103776","DOIUrl":"10.1016/j.dnarep.2024.103776","url":null,"abstract":"<div><div>To maintain genomic integrity, cells have evolved several conserved DNA damage response (DDR) pathways in response to DNA damage and stress conditions. Apurinic/apyrimidinic endonuclease 1 (APE1) exhibits AP endonuclease, 3′-5′ exonuclease, 3′-phosphodiesterase, and 3′-exoribonuclease activities and plays critical roles in the DNA repair and redox regulation of transcription. However, it remains unclear whether and how APE1 is involved in DDR pathways. In this perspective, we first updated our knowledge of APE1's functional domains and its nuclease activities and their specific associated substrates. We then summarized the newly discovered roles and mechanisms of action of APE1 in the global and nucleolar ATR-mediated DDR pathway. While the ATM-mediated DDR is well known to be activated by DNA double-strand breaks and oxidative stress, here we provided new perspectives as to how ATM DDR signaling is activated by indirect single-strand breaks (SSBs) resulting from genotoxic stress and defined SSB structures, and discuss how ATM kinase is directly activated and regulated by its activator, APE1. Together, accumulating body of new evidence supports the notion that APE1 is a master regulator protein of the ATR- and ATM-mediated DDR pathways. These new findings of APE1 in DDR signaling provide previously uncharacterized but critical functions and regulations of APE1 in genome integrity.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103776"},"PeriodicalIF":3.0,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142515218","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}
Pub Date : 2024-10-09DOI: 10.1016/j.dnarep.2024.103774
Max E. Douglas
Telomeres are protective nucleoprotein caps found at the natural ends of eukaryotic chromosomes and are crucial for the preservation of stable chromosomal structure. In cycling cells, telomeres are maintained by a multi-step process called telomere replication, which involves the eukaryotic replisome navigating a complex repetitive template tightly bound by specific proteins, before terminating at the chromosome end prior to a 5’ resection step that generates a protective 3’ overhang. In this review, we examine mechanistic aspects of the telomere replication process and consider how individual parts of this multistep event are integrated and coordinated with one-another.
{"title":"How to write an ending: Telomere replication as a multistep process","authors":"Max E. Douglas","doi":"10.1016/j.dnarep.2024.103774","DOIUrl":"10.1016/j.dnarep.2024.103774","url":null,"abstract":"<div><div>Telomeres are protective nucleoprotein caps found at the natural ends of eukaryotic chromosomes and are crucial for the preservation of stable chromosomal structure. In cycling cells, telomeres are maintained by a multi-step process called telomere replication, which involves the eukaryotic replisome navigating a complex repetitive template tightly bound by specific proteins, before terminating at the chromosome end prior to a 5’ resection step that generates a protective 3’ overhang. In this review, we examine mechanistic aspects of the telomere replication process and consider how individual parts of this multistep event are integrated and coordinated with one-another.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103774"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142483948","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}
Pub Date : 2024-10-09DOI: 10.1016/j.dnarep.2024.103773
Md Ratul Rahman, Ryotaro Kawasumi, Kouji Hirota
Remdesivir is a 1’-cyano-modified adenine nucleotide analog used for the treatment of COVID-19. Recently, the anti-carcinogenic effect of remdesivir has been also identified in human cancers. However, the impact of this drug and the mechanisms underlying the cellular tolerance to remdesivir have not been elucidated. Here, we explored DNA repair pathways responsible for the cellular tolerance to remdesivir by monitoring the sensitivity of 24 mutant DT40 cells deficient in various DNA repair pathways. We found that cells deficient in FEN1 displayed the highest sensitivity against remdesivir. Since FEN1 contributes to base excision repair (BER), we measured the cellular sensitivity to remdesivir in mutants deficient in BER and found that other BER mutants such as XRCC1−/− and PARP1−/− cells are tolerant to remdesivir, indicating that FEN1 contributes to cellular tolerance to remdesivir through roles other than BER. We observed augmented DNA damage and acute cell cycle arrest at early S-phase after remdesivir treatment in FEN1−/− cells. Moreover, the replication fork progression was significantly slowed by remdesivir in FEN1−/− cells, indicating a direct involvement of FEN1 in replication fork progression when replication is challenged by remdesivir. Since FEN1 contributes to Okazaki fragment maturation (OFM), a process ligating Okazaki fragments generated during lagging strand synthesis, we analyzed the kinetics of the repair of single-strand breaks (SSBs) in nascent DNA. Strikingly, FEN1−/− cells exhibited slowed kinetics in OFM, and remdesivir incorporation critically impaired this process in FEN1−/− cells. These results indicate that remdesivir is preferentially incorporated in Okazaki fragments leading to the failure of Okazaki fragment maturation and FEN1 plays a critical role in suppressing remdesivir-mediated DNA damage through Okazaki fragment processing. Collectively, we revealed a previously unappreciated role of FEN1 in the cellular tolerance to remdesivir.
雷米地韦是一种 1'-氰基修饰的腺嘌呤核苷酸类似物，用于治疗 COVID-19。最近，在人类癌症中也发现了雷米替韦的抗癌作用。然而，这种药物的影响以及细胞对雷米替韦耐受的机制尚未阐明。在这里，我们通过监测 24 个缺乏各种 DNA 修复途径的突变 DT40 细胞对雷米地韦的敏感性，探索了导致细胞对雷米地韦耐受性的 DNA 修复途径。我们发现，缺乏 FEN1 的细胞对雷米替韦的敏感性最高。由于FEN1有助于碱基切除修复（BER），我们测量了缺乏BER的突变体细胞对雷米地韦的敏感性，发现其他BER突变体，如XRCC1-/-和PARP1-/-细胞对雷米地韦有耐受性，这表明FEN1通过BER以外的作用促进细胞对雷米地韦的耐受性。我们观察到，FEN1-/-细胞在雷米替韦处理后，DNA损伤加剧，细胞周期在早期S期急剧停滞。此外，在 FEN1-/- 细胞中，雷米替韦明显减缓了复制叉的进展，这表明当复制受到雷米替韦的挑战时，FEN1 直接参与了复制叉的进展。由于 FEN1 有助于冈崎片段成熟（OFM）--一个连接滞后链合成过程中产生的冈崎片段的过程，我们分析了新生 DNA 中单链断裂（SSB）的修复动力学。令人震惊的是，FEN1-/-细胞在OFM中表现出缓慢的动力学，而雷米地韦的加入严重影响了FEN1-/-细胞的这一过程。这些结果表明，雷米地韦会优先掺入冈崎片段，导致冈崎片段成熟失败，而 FEN1 在通过冈崎片段处理抑制雷米地韦介导的 DNA 损伤方面起着关键作用。总之，我们揭示了 FEN1 在细胞耐受雷米替韦方面以前未被认识到的作用。
{"title":"The flap endonuclease-1 mediated maturation of Okazaki fragments is critical for the cellular tolerance to remdesivir","authors":"Md Ratul Rahman, Ryotaro Kawasumi, Kouji Hirota","doi":"10.1016/j.dnarep.2024.103773","DOIUrl":"10.1016/j.dnarep.2024.103773","url":null,"abstract":"<div><div>Remdesivir is a 1’-cyano-modified adenine nucleotide analog used for the treatment of COVID-19. Recently, the anti-carcinogenic effect of remdesivir has been also identified in human cancers. However, the impact of this drug and the mechanisms underlying the cellular tolerance to remdesivir have not been elucidated. Here, we explored DNA repair pathways responsible for the cellular tolerance to remdesivir by monitoring the sensitivity of 24 mutant DT40 cells deficient in various DNA repair pathways. We found that cells deficient in FEN1 displayed the highest sensitivity against remdesivir. Since FEN1 contributes to base excision repair (BER), we measured the cellular sensitivity to remdesivir in mutants deficient in BER and found that other BER mutants such as <em>XRCC1</em><sup><em>−/−</em></sup> and <em>PARP1</em><sup><em>−/−</em></sup> cells are tolerant to remdesivir, indicating that FEN1 contributes to cellular tolerance to remdesivir through roles other than BER. We observed augmented DNA damage and acute cell cycle arrest at early S-phase after remdesivir treatment in <em>FEN1</em><sup><em>−/−</em></sup> cells. Moreover, the replication fork progression was significantly slowed by remdesivir in <em>FEN1</em><sup><em>−/−</em></sup> cells, indicating a direct involvement of FEN1 in replication fork progression when replication is challenged by remdesivir. Since FEN1 contributes to Okazaki fragment maturation (OFM), a process ligating Okazaki fragments generated during lagging strand synthesis, we analyzed the kinetics of the repair of single-strand breaks (SSBs) in nascent DNA. Strikingly, <em>FEN1</em><sup><em>−/−</em></sup> cells exhibited slowed kinetics in OFM, and remdesivir incorporation critically impaired this process in <em>FEN1</em><sup><em>−/−</em></sup> cells. These results indicate that remdesivir is preferentially incorporated in Okazaki fragments leading to the failure of Okazaki fragment maturation and FEN1 plays a critical role in suppressing remdesivir-mediated DNA damage through Okazaki fragment processing. Collectively, we revealed a previously unappreciated role of FEN1 in the cellular tolerance to remdesivir.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103773"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142434414","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}
Pub Date : 2024-10-09DOI: 10.1016/j.dnarep.2024.103771
Rowyn C. Liebau , Crystal Waters , Arooba Ahmed , Rajesh K. Soni , Jean Gautier
DNA interstrand crosslinks (ICLs) are covalent bonds between bases on opposing strands of the DNA helix which prevent DNA melting and subsequent DNA replication or RNA transcription. Here, we show that Ultraviolet Stimulated Scaffold Protein A (UVSSA) is critical for ICL repair in human cells, at least in part via the transcription coupled ICL repair (TC-ICR) pathway. Inactivation of UVSSA sensitizes human cells to ICL-inducing drugs, and delays ICL repair. UVSSA is required for replication-independent repair of a single ICL in a fluorescence-based reporter assay. UVSSA localizes to chromatin following ICL damage, and interacts with transcribing Pol II, CSA, CSB, and TFIIH. Specifically, UVSSA interaction with TFIIH is required for ICL repair and transcription inhibition blocks localization of transcription coupled repair factors to ICL damaged chromatin. Finally, UVSSA expression positively correlates with ICL-based chemotherapy resistance in human cancer cell lines. Our data strongly suggest that UVSSA is a novel ICL repair factor functioning in TC-ICR. These results provide further evidence that TC-ICR is a bona fide ICL repair mechanism that contributes to crosslinker drug resistance independently of replication-coupled ICL repair.
{"title":"UVSSA facilitates transcription-coupled repair of DNA interstrand crosslinks","authors":"Rowyn C. Liebau , Crystal Waters , Arooba Ahmed , Rajesh K. Soni , Jean Gautier","doi":"10.1016/j.dnarep.2024.103771","DOIUrl":"10.1016/j.dnarep.2024.103771","url":null,"abstract":"<div><div>DNA interstrand crosslinks (ICLs) are covalent bonds between bases on opposing strands of the DNA helix which prevent DNA melting and subsequent DNA replication or RNA transcription. Here, we show that Ultraviolet Stimulated Scaffold Protein A (UVSSA) is critical for ICL repair in human cells, at least in part via the transcription coupled ICL repair (TC-ICR) pathway. Inactivation of UVSSA sensitizes human cells to ICL-inducing drugs, and delays ICL repair. UVSSA is required for replication-independent repair of a single ICL in a fluorescence-based reporter assay. UVSSA localizes to chromatin following ICL damage, and interacts with transcribing Pol II, CSA, CSB, and TFIIH. Specifically, UVSSA interaction with TFIIH is required for ICL repair and transcription inhibition blocks localization of transcription coupled repair factors to ICL damaged chromatin. Finally, UVSSA expression positively correlates with ICL-based chemotherapy resistance in human cancer cell lines. Our data strongly suggest that UVSSA is a novel ICL repair factor functioning in TC-ICR. These results provide further evidence that TC-ICR is a <em>bona fide</em> ICL repair mechanism that contributes to crosslinker drug resistance independently of replication-coupled ICL repair.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"143 ","pages":"Article 103771"},"PeriodicalIF":3.0,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142396247","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}
Pub Date : 2024-10-07DOI: 10.1016/j.dnarep.2024.103772
Marcos Jiménez-Juliana, María I. Martínez-Jiménez, Luis Blanco
Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized in vivo into the active form remdesivir triphosphate (RTP), an analogue of adenosine triphosphate (ATP) with a cyano group substitution in the carbon 1’ of the ribose (1’-CN). RTP is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked in vitro by RTP, but the low impact in vivo on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (HsPrimPol), which also operates in mitochondria. In this work, we have tested if RTP is a valid substrate for both DNA primase and DNA polymerase activities of HsPrimPol, and its impact in the production of mature DNA primers. RTP resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with ATP. Nevertheless, RTP-initiated primers are abortive, ocassionally reaching a maximal length of 4–5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of ATP, the natural substrate, is much higher than the intracellular concentration of RTP, it is unlikely that HsPrimPol would use RTP for primer synthesis during a remdesivir treatment in real patients.
雷米地韦是一种广谱抗病毒药物，已被批准用于治疗 COVID-19。雷米替韦实际上是一种原药，在体内代谢为活性形式雷米替韦三磷酸酯（RTP），这是一种三磷酸腺苷（ATP）的类似物，在核糖（1'-CN）的碳1'上有一个氰基取代基。RTP 是 RNA 合成的底物，很容易被病毒 RNA 依赖性 RNA 聚合酶（RdRp）结合。重要的是，雷米替韦一旦加入（现在是单磷酸），就会成为延迟链终止器，从而阻断病毒 RNA 的合成。据报道，线粒体 Polγ 在体外也会受到 RTP 的阻断，但在体内对线粒体 DNA 复制停滞的影响较小，这可能是由于同样在线粒体中运行的人类 DNA 定向 DNA 磷酸酶/聚合酶（HsPrimPol）的抑制作用。在这项工作中，我们测试了 RTP 是否是 HsPrimPol 的 DNA 引物酶和 DNA 聚合酶活性的有效底物，以及它对成熟 DNA 引物生成的影响。结果表明，RTP 是一种无效的延伸底物，但它可以与 ATP 竞争，用于在 5´site 处启动引物。尽管如此，RTP 启动的引物是无效的，偶尔会达到 4-5 个核苷酸的最大长度，并且不支持由引物/模板扭曲介导的延伸。不过，考虑到天然底物 ATP 的浓度远高于细胞内 RTP 的浓度，因此 HsPrimPol 不太可能在实际患者接受雷米替韦治疗期间使用 RTP 进行引物合成。
{"title":"Remdesivir triphosphate is a valid substrate to initiate synthesis of DNA primers by human PrimPol","authors":"Marcos Jiménez-Juliana, María I. Martínez-Jiménez, Luis Blanco","doi":"10.1016/j.dnarep.2024.103772","DOIUrl":"10.1016/j.dnarep.2024.103772","url":null,"abstract":"<div><div>Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized <em>in vivo</em> into the active form remdesivir triphosphate (<em>RTP</em>), an analogue of adenosine triphosphate (<em>ATP</em>) with a cyano group substitution in the carbon 1’ of the ribose (1’-CN). <em>RTP</em> is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked <em>in vitro</em> by <em>RTP</em>, but the low impact <em>in vivo</em> on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (<em>Hs</em>PrimPol), which also operates in mitochondria. In this work, we have tested if <em>RTP</em> is a valid substrate for both DNA primase and DNA polymerase activities of <em>Hs</em>PrimPol, and its impact in the production of mature DNA primers. <em>RTP</em> resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with <em>ATP</em>. Nevertheless, <em>RTP</em>-initiated primers are abortive, ocassionally reaching a maximal length of 4–5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of <em>ATP</em>, the natural substrate, is much higher than the intracellular concentration of <em>RTP</em>, it is unlikely that <em>Hs</em>PrimPol would use <em>RTP</em> for primer synthesis during a remdesivir treatment in real patients.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"143 ","pages":"Article 103772"},"PeriodicalIF":3.0,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142396246","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}
Pub Date : 2024-10-06DOI: 10.1016/j.dnarep.2024.103769
Calvin Shun Yu Lo , Nitika Taneja , Arnab Ray Chaudhuri
Laboratory automation and quantitative high-content imaging are pivotal in advancing diverse scientific fields. These innovative techniques alleviate the burden of manual labour, facilitating large-scale experiments characterized by exceptional reproducibility. Nonetheless, the seamless integration of such systems continues to pose a constant challenge in many laboratories. Here, we present a meticulously designed workflow that automates the immunofluorescence staining process, coupled with quantitative high-content imaging to study DNA damage signalling as an example. This is achieved by using an automatic liquid handling system for sample preparation. Additionally, we also offer practical recommendations aimed at ensuring the reproducibility and scalability of experimental outcomes. We illustrate the high level of efficiency and reproducibility achieved through the implementation of the liquid handling system but also addresses the associated challenges. Furthermore, we extend the discussion into critical aspects such as microscope selection, optimal objective choices, and considerations for high-content image acquisition. Our study streamlines the image analysis process, offering valuable recommendations for efficient computing resources and the integration of cutting-edge deep learning techniques. Emphasizing the paramount importance of robust data management systems aligned with the FAIR data principles, we provide practical insights into suitable storage options and effective data visualization techniques. Together, our work serves as a comprehensive guide for life science laboratories seeking to elevate their high-content quantitative imaging capabilities through the seamless integration of advanced laboratory automation.
实验室自动化和定量高含量成像技术在推动各科学领域的发展方面发挥着举足轻重的作用。这些创新技术减轻了人工劳动的负担，促进了以卓越的可重复性为特点的大规模实验。然而，这些系统的无缝集成仍然是许多实验室不断面临的挑战。在这里，我们以研究 DNA 损伤信号为例，介绍了一种精心设计的工作流程，它能自动完成免疫荧光染色过程，并结合定量高含量成像技术。这是通过使用自动液体处理系统进行样品制备实现的。此外，我们还提供了实用建议，旨在确保实验结果的可重复性和可扩展性。我们展示了通过实施液体处理系统实现的高效率和可重复性，同时也探讨了相关的挑战。此外，我们还将讨论扩展到显微镜选择、最佳物镜选择和高内容图像采集注意事项等关键方面。我们的研究简化了图像分析流程，为高效计算资源和尖端深度学习技术的整合提供了宝贵建议。我们强调了符合 FAIR 数据原则的强大数据管理系统的重要性，并就合适的存储选项和有效的数据可视化技术提供了实用的见解。总之，我们的工作可作为生命科学实验室的综合指南，帮助实验室通过无缝集成先进的实验室自动化技术，提升高内涵定量成像能力。
{"title":"Enhancing quantitative imaging to study DNA damage response: A guide to automated liquid handling and imaging","authors":"Calvin Shun Yu Lo , Nitika Taneja , Arnab Ray Chaudhuri","doi":"10.1016/j.dnarep.2024.103769","DOIUrl":"10.1016/j.dnarep.2024.103769","url":null,"abstract":"<div><div>Laboratory automation and quantitative high-content imaging are pivotal in advancing diverse scientific fields. These innovative techniques alleviate the burden of manual labour, facilitating large-scale experiments characterized by exceptional reproducibility. Nonetheless, the seamless integration of such systems continues to pose a constant challenge in many laboratories. Here, we present a meticulously designed workflow that automates the immunofluorescence staining process, coupled with quantitative high-content imaging to study DNA damage signalling as an example. This is achieved by using an automatic liquid handling system for sample preparation. Additionally, we also offer practical recommendations aimed at ensuring the reproducibility and scalability of experimental outcomes. We illustrate the high level of efficiency and reproducibility achieved through the implementation of the liquid handling system but also addresses the associated challenges. Furthermore, we extend the discussion into critical aspects such as microscope selection, optimal objective choices, and considerations for high-content image acquisition. Our study streamlines the image analysis process, offering valuable recommendations for efficient computing resources and the integration of cutting-edge deep learning techniques. Emphasizing the paramount importance of robust data management systems aligned with the FAIR data principles, we provide practical insights into suitable storage options and effective data visualization techniques. Together, our work serves as a comprehensive guide for life science laboratories seeking to elevate their high-content quantitative imaging capabilities through the seamless integration of advanced laboratory automation.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103769"},"PeriodicalIF":3.0,"publicationDate":"2024-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142434415","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}
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