bims-replis Biomed News
on Replisome
Issue of 2025–04–06
seven papers selected by
Anna Zawada, International Centre for Translational Eye Research



  1. bioRxiv. 2025 Mar 21. pii: 2025.03.21.644660. [Epub ahead of print]
      The two-metal ion mechanism for catalysis of RNA and DNA synthesis by 5'-3' polymerases has been extensively characterized. The 3'-5' polymerase family of enzymes, consisting of tRNA His guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), perform a similar nucleotide addition reaction, but in the reverse direction, adding Watson-Crick base paired NTPs to the 5'-ends of RNA substrates, yet the effect of divalent cations beyond magnesium has not been described. Here, we examined the effects of five divalent cations (Mg 2+ , Mn 2+ , Co 2+ , Ni 2+ and Ca 2+ ) on templated nucleotide addition activity and kinetics of 5'-activation by ATP catalyzed by recombinantly purified, metal-free TLPs from organisms from diverse domains of life. This work revealed that different TLPs exhibit distinct dependencies on the concentration and identity of divalent metal ions that support effective catalysis. The patterns of metal ion usage demonstrated here for TLPs evince features that are characteristic of both canonical 5'-3' polymerases and DNA/RNA ligases. Similar to 5'-3' polymerases, some metals were also seen to be mutagenic in the context of TLP catalysis. Furthermore, we provide the first direct evidence that both ATP and the NTP poised for nucleotidyl transfer are present in the active site during the 5'-adenylylation. These results provide the first in-depth study of the role of the two-metal ion mechanism in TLP catalysis that was first suggested by structures of these enzymes.
    DOI:  https://doi.org/10.1101/2025.03.21.644660
  2. PLoS Genet. 2025 Apr 01. 21(4): e1011405
      The integrity of the replication forks is essential for an accurate and timely completion of genome duplication. However, little is known about how cells deal with broken replication forks. We have generated in yeast a system based on a chimera of the largest subunit of the ssDNA binding complex RPA fused to the micrococcal nuclease (Rfa1-MN) to induce double-strand breaks (DSBs) at replication forks and searched for mutants affected in their repair. Our results show that the core homologous recombination (HR) proteins involved in the formation of the ssDNA/Rad51 filament are essential for the repair of DSBs at forks, whereas non-homologous end joining plays no role. Apart from the endonucleases Mus81 and Yen1, the repair process employs fork-associated HR factors, break-induced replication (BIR)-associated factors and replisome components involved in sister chromatid cohesion and fork stability, pointing to replication fork restart by BIR followed by fork restoration. Notably, we also found factors controlling the length of G1, suggesting that a minimal number of active origins facilitates the repair by converging forks. Our study has also revealed a requirement for checkpoint functions, including the synthesis of Dun1-mediated dNTPs. Finally, our screening revealed minimal impact from the loss of chromatin factors, suggesting that the partially disassembled nucleosome structure at the replication fork facilitates the accessibility of the repair machinery. In conclusion, this study provides an overview of the factors and mechanisms that cooperate to repair broken forks.
    DOI:  https://doi.org/10.1371/journal.pgen.1011405
  3. bioRxiv. 2025 Mar 18. pii: 2025.03.18.643935. [Epub ahead of print]
      Methylglyoxal (MG) is a highly reactive aldehyde that is produced endogenously during metabolism and is derived from exogenous sources such as sugary food items and cigarette smoke. Unless detoxified by glyoxalases (Glo1 and Glo2), MG can readily react with all major biomolecules, including DNA and proteins, generating characteristic lesions and glycation-derived by- products. As a result, MG exposure has been linked to a variety of human diseases, including cancers. Prior studies show that MG can glycate DNA, preferentially on guanine residues, and cause DNA damage. However, the mutagenicity of MG is poorly understood in vivo. In the context of cancer, it is essential to comprehend the true contribution of MG to genome instability and global mutational burden. In the present study, we show that MG can robustly mutagenize induced single-stranded DNA (ssDNA) in yeast, within a guanine centered mutable motif. We demonstrate that genome-wide MG mutagenesis in ssDNA is greatly elevated throughout the genome in the absence of Glo1, and abrogated in the presence of the aldehyde quencher aminoguanidine. We uncovered strand slippage and mispairing as the predominant mechanism for generation of all MG-associated mutations, and demonstrate that the translesion polymerase Rev1 is necessary in this pathway. Finally, we find that the primary MG-associated mutation is enriched in a variety of sequenced tumor datasets. We discuss the genomic impact of methylglyoxal exposure in the context of mutagenesis, DNA damage, and carcinogenesis.
    DOI:  https://doi.org/10.1101/2025.03.18.643935
  4. Proc Natl Acad Sci U S A. 2025 Apr 08. 122(14): e2502277122
      In eukaryotes, the origin recognition complex (ORC) promotes the assembly of minichromosome maintenance 2 to 7 complexes into a head-to-head double hexamer at origin DNA in a process known as replication licensing. In this study, we present a series of cryoelectron microscopy structures of yeast ORC mutants in complex with origin DNA. We show that Orc6, the smallest subunit of ORC, utilizes its transcription factor II B-B domain to orchestrate the sequential binding of ORC to origin DNA. In addition, Orc6 plays the role of a scaffold by stabilizing the basic patch (BP) of Orc5 for ORC to capture and bend origin DNA. Importantly, disrupting DNA bending through mutating three key residues in Orc5-BP impairs ORC's ability to promote replication initiation at two points during the pre-RC assembly process. This study dissects the multifaceted role of Orc6 in orchestrating ORC's activities on DNA and underscores the vital role of DNA bending by ORC in replication licensing.
    Keywords:  DNA bending; MCM double hexamer; eukaryotic DNA replication; origin recognition complex; replication initiation
    DOI:  https://doi.org/10.1073/pnas.2502277122
  5. bioRxiv. 2025 Mar 11. pii: 2025.03.10.642433. [Epub ahead of print]
      Ring-shaped sliding clamp proteins are essential components of the replication machinery, the replisome, across all domains of life. In bacteria, DNA polymerases bind the sliding clamp, DnaN, through conserved short peptide sequences called clamp-binding motifs. Clamp binding increases the processivity and rate of DNA synthesis and is generally required for polymerase activity. The current understanding of clamp-polymerase interactions was elucidated in the model bacterium Escherichia coli , which has a single replicative polymerase, Pol III. However, many bacteria have two essential replicative polymerases, such as PolC and DnaE in Bacillus subtilis . PolC performs the bulk of DNA synthesis whereas the error-prone DnaE only synthesizes short stretches of DNA on the lagging strand. How the clamp interacts with the two polymerases and coordinates their activity is unknown. We investigated this question by combining in vivo single-molecule fluorescence microscopy with biochemical and microbiological assays. We found that PolC-DnaN binding is essential for replication, although weakening the interaction is tolerated with only minimal effects. In contrast, the DnaE-DnaN interaction is dispensable for replication. Altering the clamp-binding strength of DnaE produces only subtle effects on DnaE cellular localization and dynamics, but it has a substantial impact on mutagenesis. Our results support a model in which DnaE acts distributively during replication but can be stabilized on the DNA template by clamp binding. This study provides new insights into the coordination of multiple replicative polymerases in bacteria and the role of the clamp in polymerase processivity, fidelity, and exchange.
    DOI:  https://doi.org/10.1101/2025.03.10.642433
  6. In Silico Pharmacol. 2025 ;13(1): 49
      The Mpox DNA polymerase (DNA pol) plays a crucial role in the viral replication process, making it an ideal target for antiviral therapies. It facilitates the synthetic process of viral DNA, which is an integral stage in the life of a virus. The inhibition of the operation of Mpox DNA pol would interfere with the multiplication of the virus and help manage the disease. Peptides have emerged as a possible therapeutic alternative against viruses due to their distinct characteristics. Peptides have broad-spectrum antiviral activity, being effective against a variety of viruses. Using computational techniques, we attempted to explore the molecular details of the interaction between antiviral peptides and Mpox DNA pol. Two databases of antiviral peptides were screened in this study. This study used molecular docking, followed by molecular dynamics (MD) simulation and post-simulation binding energy predictions. From the 19 selected peptides with activity against DNA polymerases, two peptides-DRAVPe01393 and DRAVPe01399-were identified as particularly promising candidates. These peptides exhibited stable interactions with Mpox DNA pol and demonstrated good cell penetration potential as evident from the MD simulation studies. Notably, the peptides DRAVPe01399 and DRAVPe01393 have a better binding affinity of - 60.86 kcal/mol and - 47.92 kcal/mol respectively than the control ligand Cidofovir diphosphate (- 10.79 kcal/mol). These findings could lead to the development of innovative antiviral treatments to prevent monkeypox, helping global efforts to battle this emerging infectious disease.
    Keywords:  Antiviral peptide; MD simulation; Molecular docking; Mpox DNA pol; Polymerase inhibitor
    DOI:  https://doi.org/10.1007/s40203-025-00342-4
  7. Curr Biol. 2025 Mar 27. pii: S0960-9822(25)00294-5. [Epub ahead of print]
      In bacteria, commitment to genome replication (initiation) is intricately linked to nutrient availability. Whether growth conditions affect other stages of replication beyond initiation remains to be systematically studied. To address this, we assess the replication dynamics of Caulobacter crescentus, a bacterium that undergoes only a single round of replication per cell cycle, by tracking the replisome across various growth phases and nutrient conditions. We find that the replication elongation rates slow down as cells transition from exponential (high-nutrient) to stationary (low-nutrient) phase, and this contributes significantly to the overall cell-cycle delay. Although elongation rates are correlated with growth rates, both properties are differentially influenced by nutrient status. This slowdown in replication progression is reversed via supplementation with dNTPs and is not associated with increased mutagenesis or upregulation of the DNA damage responses. We conclude that growth conditions not only dictate the commitment to replication but also the rates of genome duplication. Such regulation appears to be distinct from stress-induced replication slowdown and likely serves as an adaptive mechanism to cope with fluctuations in nutrient availability in the environment.
    Keywords:  Caulobacter crescentus; DNA replication; live-cell imaging; replication regulation; replisome dynamics; stationary phase
    DOI:  https://doi.org/10.1016/j.cub.2025.03.009