bims-replis Biomed News
on Replisome
Issue of 2025–05–04
twelve papers selected by
Anna Zawada, International Centre for Translational Eye Research
  1. The proofreading mechanism of the human leading strand DNA polymerase ϵ holoenzyme.
  2. Replicative DNA polymerase epsilon and delta holoenzymes show wide-ranging inhibition at G-quadruplexes in the human genome.
  3. Real-time imaging of rotation during synthesis by the replisome.
  4. DNA replication in primary hepatocytes without the six-subunit ORC.
  5. Always on the Move: Overview on Chromatin Dynamics within Nuclear Processes.
  6. Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors.
  7. Replication Factor C Subunit 4 Plays a Role in Human Breast Cancer Cell Progression.
  8. Structural basis of multitasking by the apicoplast DNA polymerase from Plasmodium falciparum.
  9. Ramping up mitochondrial DNA replication.
  10. NUDT16 enhances the resistance of cancer cells to DNA-damaging agents by regulating replication fork stability via reversing HMGA1 ADP-ribosylation.
  11. Ancient Host-Virus Gene Transfer Hints at a Diverse Pre-LECA Virosphere.
  12. OriV-Finder: a comprehensive web server for bacterial plasmid replication origin analysis.
  1. bioRxiv. 2025 Apr 12. pii: 2025.04.11.648458. [Epub ahead of print]
    The proofreading mechanism of the human leading strand DNA polymerase ϵ holoenzyme.
    Feng Wang, Qing He, Michael E O'Donnell, Huilin Li.
      The eukaryotic leading strand DNA polymerase epsilon is a dual function enzyme with a proofreading exonuclease site located 40 angstroms from the DNA synthesizing polymerase site. Errors in Pol epsilon proofreading can cause various mutations, including C to G transversions, the most prevalent mutation in cancers and genetic diseases. Pol epsilon interacts with all three subunits of the PCNA ring to assemble a functional holoenzyme. Despite previous studies on proofreading of several polymerases, how Pol epsilon, or any Pol complexed with its sliding clamp proofreads a mismatch generated in situ has been unknown. We show here by cryo-EM that a template/primer DNA substrate with a pre-existing mismatch cannot enter the exo site of Pol epsilon/PCNA holoenzyme, but a mismatch generated in the Pol site yields three proofreading intermediates of Pol epsilon/PCNA holoenzyme. These intermediates reveal how the mismatch is dislodged from the Pol site, how the DNA unwinds 6 base pairs and how the unpaired primer 3'-end is inserted into the exo site for cleavage. These results unexpectedly demonstrate that PCNA imposes strong steric constraints that extend unwinding and direct the trajectory of mismatched DNA, and that this trajectory is dramatically different than for Pol epsilon in the absence of PCNA. These findings suggest a physiologically relevant proofreading mechanism for the human Pol epsilon/PCNA holoenzyme.
    DOI:  https://doi.org/10.1101/2025.04.11.648458
  2. Nucleic Acids Res. 2025 Apr 22. pii: gkaf352. [Epub ahead of print]53(8):
    Replicative DNA polymerase epsilon and delta holoenzymes show wide-ranging inhibition at G-quadruplexes in the human genome.
    Suzanne E Hile, Matthias H Weissensteiner, Kara G Pytko, Joseph Dahl, Eduard Kejnovsky, Iva Kejnovská, Mark Hedglin, Ilias Georgakopoulos-Soares, Kateryna D Makova, Kristin A Eckert.
      G-quadruplexes (G4s) are functional elements of the human genome, some of which inhibit DNA replication. We investigated replication of G4s within highly abundant microsatellite (GGGA, GGGT) and transposable element (L1 and SVA) sequences. We found that genome-wide, numerous motifs are located preferentially on the replication leading strand and the transcribed strand templates. We directly tested replicative polymerase ϵ and δ holoenzyme inhibition at these G4s, compared to low abundant motifs. For all G4s, DNA synthesis inhibition was higher on the G-rich than C-rich strand or control sequence. No single G4 was an absolute block for either holoenzyme; however, the inhibitory potential varied over an order of magnitude. Biophysical analyses showed the motifs form varying topologies, but replicative polymerase inhibition did not correlate with a specific G4 structure. Addition of the G4 stabilizer pyridostatin severely inhibited forward polymerase synthesis specifically on the G-rich strand, enhancing G/C strand asynchrony. Our results reveal that replicative polymerase inhibition at every G4 examined is distinct, causing complementary strand synthesis to become asynchronous, which could contribute to slowed fork elongation. Altogether, we provide critical information regarding how replicative eukaryotic holoenzymes navigate synthesis through G4s naturally occurring thousands of times in functional regions of the human genome.
    DOI:  https://doi.org/10.1093/nar/gkaf352
  3. bioRxiv. 2025 Apr 07. pii: 2025.04.01.646591. [Epub ahead of print]
    Real-time imaging of rotation during synthesis by the replisome.
    Thomas M Retzer, Lional T Rajappa, Masateru Takahashi, Samir M Hamdan, Karl E Duderstadt.
      During chromosome replication, unwinding by the helicase and synthesis by the polymerases can lead to overwinding and supercoiling of DNA. The mechanical consequences of these events and resulting local dynamics at the replication fork are not well understood. To address these issues, we developed a transverse DNA flow-stretching approach to spatially resolve the parental, leading and lagging strands in real-time. Using bacteriophage T7 as a model system, this approach revealed bursts of high-speed replisome rotation that support continuous DNA synthesis. Surprisingly, excessive rotation does not reduce replisome speed, but increases pausing, reduces processivity, and increases polymerase exchange. Taken together, our observations reveal intrinsic pathways to overcome challenges posed by unfavorable DNA topologies during DNA replication.
    DOI:  https://doi.org/10.1101/2025.04.01.646591
  4. Elife. 2025 Apr 30. pii: RP102915. [Epub ahead of print]13
    DNA replication in primary hepatocytes without the six-subunit ORC.
    Róża K Przanowska, Yuechuan Chen, Takayuki-Okano Uchida, Etsuko Shibata, Xiaoxiao Hao, Isaac Segura Rueda, Kate Jensen, Piotr Przanowski, Anthony Trimboli, Yoshiyuki Shibata, Gustavo Leone, Anindya Dutta.
      The six-subunit ORC is essential for the initiation of DNA replication in eukaryotes. Cancer cell lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2, or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.
    Keywords:  cell biology; genetics; genomics; mouse; mouse embryo fibroblasts; mouse liver; primary hepatocytes
    DOI:  https://doi.org/10.7554/eLife.102915
  5. Biochemistry. 2025 May 01.
    Always on the Move: Overview on Chromatin Dynamics within Nuclear Processes.
    Charlotte M Delvaux de Fenffe, Jolijn Govers, Francesca Mattiroli.
      Our genome is organized into chromatin, a dynamic and modular structure made of nucleosomes. Chromatin organization controls access to the DNA sequence, playing a fundamental role in cell identity and function. How nucleosomes enable these processes is an active area of study. In this review, we provide an overview of chromatin dynamics, its properties, mechanisms, and functions. We highlight the diverse ways by which chromatin dynamics is controlled during transcription, DNA replication, and repair. Recent technological developments have promoted discoveries in this area, to which we provide an outlook on future research directions.
    Keywords:  DNA sequence; chromatin dynamics; chromatin organization; nucleosome
    DOI:  https://doi.org/10.1021/acs.biochem.5c00114
  6. Cell. 2025 Apr 24. pii: S0092-8674(25)00414-3. [Epub ahead of print]
    Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors.
    Xing Kang, Xinran Li, Jiaqi Zhou, Yang Zhang, Lingyu Qiu, Congcong Tian, Zhiwen Deng, Xiaoyan Liang, Ziwei Zhang, Songlin Du, Suili Hu, Nan Wang, Zhen Yue, Yajing Xu, Yuan Gao, Junbiao Dai, Zhiquan Wang, Chuanhe Yu, Jinyi Chen, Yuchun Wu, Liangming Chen, Yuan Yao, Sitong Yao, Xinran Yang, Lixia Yan, Qing Wen, Olivia M Depies, Kuiming Chan, Xiaohuan Liang, Gang Li, Zhike Zi, Xiangyu Liu, Haiyun Gan.
      Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells. However, the functional significance of ecDNA and the molecular components involved in its replication and maintenance remain largely unknown. Here, using CRISPR-C technology, we generated ecDNA-carrying (ecDNA+) cell models. By leveraging these models alongside other well-established systems, we demonstrated that ecDNA can replicate and be maintained in ecDNA+ cells. The replication of ecDNA activates the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) pathway. Topoisomerases, such as TOP1 and TOP2B, play a role in ecDNA replication-induced DNA double-strand breaks (DSBs). A subset of these elevated DSBs persists into the mitotic phase and is primarily repaired by the alternative non-homologous end joining (alt-NHEJ) pathway, which involves POLθ and LIG3. Correspondingly, ecDNA maintenance requires DDR, and inhibiting DDR impairs the circularization of ecDNA. In summary, we demonstrate reciprocal interactions between ecDNA maintenance and DDR, providing new insights into the detection and treatment of ecDNA+ tumors.
    Keywords:  DNA damage response; LIG3; TOP1; TOP2B; alt-NHEJ; ecDNA
    DOI:  https://doi.org/10.1016/j.cell.2025.04.012
  7. Cancer Genomics Proteomics. 2025 May-Jun;22(3):22(3): 478-490
    Replication Factor C Subunit 4 Plays a Role in Human Breast Cancer Cell Progression.
    Jae Woong Koh, Seon-Joo Park.
       BACKGROUND/AIM: Breast cancer is a heterogenous disease characterized by complex molecular pathways that drive its progression. Despite advances in treatment strategies, the need for novel therapeutic targets remains critical. Replication factor C subunit 4 (RFC4) is an important component of the DNA replication machinery and repair pathways. Its precise regulation ensures genomic stability and its dysregulation is implicated in various cancers. However, its oncogenic role in breast cancer is unclear. Therefore, this study aimed to elucidate the biological role of RFC4 in breast cancer.
    MATERIALS AND METHODS: Breast cancer cell lines MCF7, BT-549, and MDA-MB-231 were transfected with control and RFC4 siRNA to investigate biological functions of RFC4 in breast cancer. Cell proliferation was measured using the MTT and colony formation assays. In addition, cell cycle analysis, migration, and invasion assays were performed on RFC4-depleted breast cancer cells.
    RESULTS: siRNA-mediated RFC4 knockdown inhibited breast cancer cell proliferation and cell cycle arrest, and reduced cell migration and invasion ability.
    CONCLUSION: Our findings highlight the critical role of RFC4 in breast cancer progression. The observed decrease in cell proliferation and clonogenic potential following RFC4 knockdown suggests its potential as a therapeutic target for breast cancer.
    Keywords:  RFC4; breast cancer; cell cycle; invasion; migration; proliferation
    DOI:  https://doi.org/10.21873/cgp.20515
  8. bioRxiv. 2025 Apr 09. pii: 2025.04.09.647933. [Epub ahead of print]
    Structural basis of multitasking by the apicoplast DNA polymerase from Plasmodium falciparum.
    Anamika Kumari, Theodora Enache, Timothy D Craggs, Janice D Pata, Indrajit Lahiri.
      Plasmodium falciparum is a unicellular eukaryotic pathogen responsible for the majority of malaria-related fatalities. Plasmodium belongs to the phylum Apicomplexa and like most members of this phylum, contains a non-photosynthetic plastid called the apicoplast. The apicoplast has its own genome, which is replicated by a dedicated apicoplast replisome. Unlike other cellular replisomes, the apicoplast replisome uses a single DNA polymerase (apPol) for copying the apicoplast DNA. Being the only DNA polymerase in the apicoplast, apPol is expected to multitask, catalysing both replicative and lesion bypass synthesis. Replicative synthesis typically relies on a restrictive active site for high accuracy while lesion bypass requires an open active site. This raises the question how does apPol combine the structural features of multiple DNA polymerases in a single protein. Using single particle electron cryomicroscopy (cryoEM), we have solved the structures of apPol bound to its DNA and nucleotide substrates in five pre-chemistry conformational states, allowing us to describe the events leading up to nucleotide incorporation and answer how apPol incorporates features of multiple polymerases. We found that, unlike most replicative polymerases, apPol can accommodate a nascent base pair with the fingers in an open configuration, which might facilitate the lesion bypass activity. In the fingers open state we identified a nascent base pair checkpoint that can preferentially select a Watson-Crick base pair, an essential requirement for replicative synthesis. Taken together these structural features explain how apPol may balance replicative and lesion bypass synthesis.
    DOI:  https://doi.org/10.1101/2025.04.09.647933
  9. Nat Rev Drug Discov. 2025 May 02.
    Ramping up mitochondrial DNA replication.
    Katie Kingwell.
      
    DOI:  https://doi.org/10.1038/d41573-025-00079-x
  10. J Biol Chem. 2025 Apr 25. pii: S0021-9258(25)00400-4. [Epub ahead of print] 108551
    NUDT16 enhances the resistance of cancer cells to DNA-damaging agents by regulating replication fork stability via reversing HMGA1 ADP-ribosylation.
    Yingshi Zhou, Zhihuai Deng, Shiyu Xiong, Wenjia Li, Wanrong Luo, Man Luo, Haifeng Tang, Wenjing Wu, Carmen Chak-Lui Wong, Dong Yin, Kaishun Hu, Baoming Luo.
      Precise DNA replication is the basis for maintaining cell proliferation and genome stability. Current chemotherapy drugs and radiotherapy induce cell death by aggravating replication stress, albeit with poor efficacy. The replication stress response has been shown to play fundamental roles in resistance to radiotherapy and chemotherapy. High mobility group A1 (HMGA1) promotes tumor progression by regulating autophagy, angiogenesis, and chemoresistance; however, its role in coordinating replication stress and cell cycle progression remains elusive. Our results indicated that HMGA1 recruited FANCD2 to promote DNA replication and cell cycle progression both by attenuating R-loop-induced replication stress and by protecting stalled replication forks from degradation, ultimately enhancing tumor resistance to chemotherapy and irradiation (IR) treatment. We also identified HMGA1 as a novel substrate for the dePARylase NUDT16. NUDT16 was found to suppress the binding of HMGA1 to the E3 ubiquitin ligase CHFR by removing its PARylation at Glu 50, thereby reducing its ubiquitin-proteasome pathway-mediated degradation and enhancing HMGA1 protein stability. NUDT16-HMGA1 inhibition can significantly improve the sensitivity of tumor cells to chemotherapy and IR treatment. Collectively, these data suggest that NUDT16 enhances the ability of tumor cells to cope with replication stress by reversing the PARylation and positively regulating the protein expression of HMGA1. Therefore, targeting the NUDT16-HMGA1 pathway may be a novel strategy to enhance the sensitivity of radiotherapy and chemotherapy.
    Keywords:  HMGA1; NUDT16; PARylation; replication stress; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2025.108551
  11. J Mol Evol. 2025 Apr 29.
    Ancient Host-Virus Gene Transfer Hints at a Diverse Pre-LECA Virosphere.
    Sangita Karki, Zachary K Barth, Frank O Aylward.
      The details surrounding the early evolution of eukaryotes and their viruses are largely unknown. Several key enzymes involved in DNA synthesis and transcription are shared between eukaryotes and large DNA viruses in the phylum Nucleocytoviricota, but the evolutionary relationships between these genes remain unclear. In particular, previous studies of eukaryotic DNA and RNA polymerases often show deep-branching clades of eukaryotes and viruses indicative of ancient gene exchange. Here, we performed updated phylogenetic analysis of eukaryotic and viral family B DNA polymerases, multimeric RNA polymerases, and mRNA-capping enzymes to explore their evolutionary relationships. Our results show that viral enzymes form clades that are typically adjacent to eukaryotes, suggesting that they originate prior to the emergence of the Last Eukaryotic Common Ancestor (LECA). The machinery for viral DNA replication, transcription, and mRNA capping are all key processes needed for the maintenance of virus factories, which are complex structures formed by many nucleocytoviruses during infection, indicating that viruses capable of making these structures are ancient. These findings hint at a diverse and complex pre-LECA virosphere and indicate that large DNA viruses may encode proteins that are relics of extinct proto-eukaryotic lineages.
    Keywords:  Early eukaryotes; Giant viruses; Mirusviruses; Nucleocytoviricota; Virus factory
    DOI:  https://doi.org/10.1007/s00239-025-10246-8
  12. Nucleic Acids Res. 2025 Apr 29. pii: gkaf341. [Epub ahead of print]
    OriV-Finder: a comprehensive web server for bacterial plasmid replication origin analysis.
    Yujie Li, Feng Gao.
      To rapidly identify and systematically analyse the vegetative replication origins (oriVs) of bacterial plasmids, we present OriV-Finder, a comprehensive web server for bacterial plasmid replication origin analysis. To fulfil this purpose, we collected 470 replication initiation proteins (RIPs) reported in the literature, identified 35 conserved domains associated with RIPs, and summarized conserved features of oriVs for various replication initiation mechanisms. Therefore, OriV-Finder could accurately identify the homologous genes of RIPs and then assess the likelihood of each intergenic sequence as a potential oriV based on the information of RIPs and conserved features. Consequently, the potential oriVs could be designated using a priority-based scoring system. As a user-friendly web server, OriV-Finder integrates visualization modules of oriVs, RIPs, and genomes, which facilitates the analysis and validation of oriVs. OriV-Finder is freely available to all users without any login requirement at https://tubic.org/OriV-Finder/.
    DOI:  https://doi.org/10.1093/nar/gkaf341
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