The Repeat Expansion Diseases (REDs) are a large group of human genetic disorders that result from an increase in the number of repeats in a disease-specific tandem repeat or microsatellite. Emerging evidence suggests that the repeats trigger an error-prone form of DNA repair that causes the expansion mutation by exploiting a limitation in normal mismatch repair. Furthermore, while much remains to be understood about how the mutation causes pathology in different diseases in this group, there is evidence to suggest that some of the downstream consequences of repeat expansion trigger the DNA damage response in ways that contribute to disease pathology. This review will discuss these subjects in the context of the Fragile X-related disorders (aka the FMR1 disorders) that provide a particularly interesting example of the intersection between the repeats and the DNA damage response that may also be relevant for many other diseases in this group.
重复扩增疾病（REDs）是一大类人类遗传疾病，是由于疾病特异性串联重复或微卫星的重复次数增加而导致的。新的证据表明，这些重复序列会引发一种容易出错的 DNA 修复形式，从而利用正常错配修复的局限性导致扩增突变。此外，尽管人们对突变如何导致该组不同疾病的病理变化仍有很多不解，但有证据表明，重复扩增的一些下游后果会触发 DNA 损伤反应，从而导致疾病的病理变化。本综述将以脆性 X 相关疾病（又称 FMR1 相关疾病）为背景讨论这些问题，该疾病提供了一个特别有趣的例子，说明了重复与 DNA 损伤反应之间的交叉关系，而这种交叉关系可能也与这类疾病中的许多其他疾病有关。
{"title":"Intersection of the fragile X-related disorders and the DNA damage response","authors":"Daman Kumari , Jessalyn Grant-Bier , Farid Kadyrov , Karen Usdin","doi":"10.1016/j.dnarep.2024.103785","DOIUrl":"10.1016/j.dnarep.2024.103785","url":null,"abstract":"<div><div>The Repeat Expansion Diseases (REDs) are a large group of human genetic disorders that result from an increase in the number of repeats in a disease-specific tandem repeat or microsatellite. Emerging evidence suggests that the repeats trigger an error-prone form of DNA repair that causes the expansion mutation by exploiting a limitation in normal mismatch repair. Furthermore, while much remains to be understood about how the mutation causes pathology in different diseases in this group, there is evidence to suggest that some of the downstream consequences of repeat expansion trigger the DNA damage response in ways that contribute to disease pathology. This review will discuss these subjects in the context of the Fragile X-related disorders (aka the <em>FMR1</em> disorders) that provide a particularly interesting example of the intersection between the repeats and the DNA damage response that may also be relevant for many other diseases in this group.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103785"},"PeriodicalIF":3.0,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142644799","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 : 2024-11-07DOI: 10.1016/j.dnarep.2024.103784
Qinwei Zhu, Xuening Chen, Zhonghui Lin
Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.
基质抗原 1 和 2（STAG1 和 STAG2）是凝聚素复合物的两个互斥成分，对中心粒和端粒的凝聚至关重要。除了其结构作用外，STAG2 还在同源重组（HR）修复中发挥着关键作用，并已成为癌症治疗中一个很有前景的治疗靶点。在这里，我们采用了基于荧光偏振（FP）的高通量筛选方法，发现 KPT-6566 是 STAG1 和 STAG2 的双重抑制剂。生化和生物物理分析表明，KPT-6566 能直接与 STAG1 和 STAG2 结合，破坏它们与 SCC1 和双链 DNA 的相互作用。转移期染色体扩散试验表明，KPT-6566 会导致 HeLa 细胞中染色体过早分离并诱发染色体损伤。此外，KPT-6566 还会损害 DNA 损伤修复，导致双链断裂积累和细胞凋亡。最后，KPT-6566 能使 HeLa 和 HepG2 细胞对 PARP 抑制剂 Olaparib 和 NHEJ 抑制剂 UMI-77 敏感，在抑制细胞增殖方面表现出协同效应。我们的研究结果凸显了 STAG1/2 作为癌症治疗靶点的潜力，尤其是当它们与其他 DNA 损伤反应抑制剂联合使用时。
{"title":"Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro","authors":"Qinwei Zhu, Xuening Chen, Zhonghui Lin","doi":"10.1016/j.dnarep.2024.103784","DOIUrl":"10.1016/j.dnarep.2024.103784","url":null,"abstract":"<div><div>Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103784"},"PeriodicalIF":3.0,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634202","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 : 2024-11-02DOI: 10.1016/j.dnarep.2024.103781
Zhiyu Lu , Dong Chen , Ning Zhang , Zhiyuan Zheng , Zimo Zhou , Guochen Liu , Jiawei An , Yong Wang , Yongping Su , Wensheng Chen , Fengchao Wang
DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS-1 pretreatment could increase the survival and regeneration of irradiated intestinal organoids in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6 Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms.
{"title":"Transient HR enhancement by RAD51-stimulatory compound confers protection on intestinal rather than hematopoietic tissue against irradiation in mice","authors":"Zhiyu Lu , Dong Chen , Ning Zhang , Zhiyuan Zheng , Zimo Zhou , Guochen Liu , Jiawei An , Yong Wang , Yongping Su , Wensheng Chen , Fengchao Wang","doi":"10.1016/j.dnarep.2024.103781","DOIUrl":"10.1016/j.dnarep.2024.103781","url":null,"abstract":"<div><div>DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS-1 pretreatment could increase the survival and regeneration of irradiated intestinal organoids in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6 Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms.</div></div>","PeriodicalId":300,"journal":{"name":"DNA Repair","volume":"144 ","pages":"Article 103781"},"PeriodicalIF":3.0,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634368","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 : 2024-10-26DOI: 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 : 2024-10-09DOI: 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}
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