bims-replis Biomed News
on Replisome
Issue of 2025–04–27
six papers selected by
Anna Zawada, International Centre for Translational Eye Research
  1. Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes.
  2. Update on R-loops in genomic integrity: Formation, functions, and implications for human diseases.
  3. HU promotes higher order chromosome organization and influences DNA replication rates in Streptococcus pneumoniae.
  4. DNA Transactions in Bacteria and Membranes: A Place for the Hfq Protein?
  5. Preparation for mitosis requires gradual CDK1 activation.
  6. RIF1 controls replication timing in early mouse embryos independently of lamina-associated nuclear organization.
  1. J Vis Exp. 2025 Apr 04.
    Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes.
    Nicholas Kusi-Appauh, Stefan H Mueller, Stephen F Ralph, Olga Yurieva, Michael E O'Donnell, Jacob S Lewis, Lisanne M Spenkelink.
      The ability of proteins involved in eukaryotic DNA replication to overcome obstacles - such as protein and DNA 'roadblocks' - is critical for ensuring faithful genome duplication. G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase δ, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.
    DOI:  https://doi.org/10.3791/68080
  2. Genes Dis. 2025 Jul;12(4): 101401
    Update on R-loops in genomic integrity: Formation, functions, and implications for human diseases.
    Min Zhu, Xinyu Wang, Hongchang Zhao, Zhenjie Wang.
      R-loops, three-strand nucleic acid structures, have emerged as crucial players in various physiological processes, including the regulation of gene expression, DNA replication, and class switch recombination. However, their presence also poses a significant threat to genome stability. A particularly challenging aspect is understanding the dynamic balance between R-loops' "light" and "dark" sites, especially concerning maintaining genome integrity. The complex and multifaceted roles of R-loops in genome stability necessitate a deeper understanding. This review offers a comprehensive exploration of the formation, resolution, and implications of R-loops, particularly in the context of DNA damage and human disease. We delve into the dualistic nature of R-loops, highlighting their role in DNA damage response and repair, and discuss the therapeutic potential arising from our evolving understanding of these enigmatic entities. Emphasizing recent advancements and unresolved questions, this review aims to provide a cohesive overview of R-loops, inviting further inquiry and investigation into their complex biological significance.
    Keywords:  DNA repair; Double strand breaks; Genome instability; Immune response; R-loops; Replication stress
    DOI:  https://doi.org/10.1016/j.gendis.2024.101401
  3. Nucleic Acids Res. 2025 Apr 22. pii: gkaf312. [Epub ahead of print]53(8):
    HU promotes higher order chromosome organization and influences DNA replication rates in Streptococcus pneumoniae.
    Maria-Vittoria Mazzuoli, Renske van Raaphorst, Louise S Martin, Florian P Bock, Agnès Thierry, Martial Marbouty, Barbora Waclawiková, Jasper Stinenbosch, Romain Koszul, Jan-Willem Veening.
      Nucleoid-associated proteins (NAPs) are crucial for maintaining chromosomal compaction and architecture, and are actively involved in DNA replication, recombination, repair, and gene regulation. In Streptococcus pneumoniae, the role of the highly conserved NAP HU in chromosome conformation has not yet been investigated. Here, we use a multi-scale approach to explore HU's role in chromosome conformation and segregation dynamics. By combining superresolution microscopy and whole-genome-binding analysis, we describe the nucleoid as a dynamic structure where HU binds transiently across the entire nucleoid, with a preference for the origin of replication over the terminus. Reducing cellular HU levels impacts nucleoid maintenance and disrupts nucleoid scaling with cell size, similar to the distortion caused by fluoroquinolones, supporting its requirement for maintaining DNA supercoiling. Furthermore, in cells lacking HU, the replication machinery is misplaced, preventing cells from initiating and proceeding with ongoing replication. Chromosome conformation capture coupled to deep sequencing (Hi-C) revealed that HU is required to maintain cohesion between the two chromosomal arms, similar to the structural maintenance of chromosome complex. Together, we show that by promoting long-range chromosome interactions and supporting the architecture of the domain encompassing the origin, HU is essential for chromosome integrity and the intimately related processes of replication and segregation.
    DOI:  https://doi.org/10.1093/nar/gkaf312
  4. Membranes (Basel). 2025 Apr 01. pii: 103. [Epub ahead of print]15(4):
    DNA Transactions in Bacteria and Membranes: A Place for the Hfq Protein?
    Sylwia Bloch, Richard R Sinden, Frank Wien, Grzegorz Węgrzyn, Véronique Arluison.
      DNA metabolism consists of crucial processes occurring in all living cells. These processes include various transactions, such as DNA replication, genetic recombination, transposition, mutagenesis, and DNA repair. While it was initially assumed that these processes might occur in the cytoplasm of prokaryotic cells, subsequent reports indicated the importance of the cell membrane in various DNA transactions. Furthermore, newly identified factors play significant roles in regulating DNA-related cellular processes. One such factor is the Hfq protein, originally discovered as an RNA chaperone but later shown to be involved in several molecular mechanisms. These include DNA transactions and interaction with the cell membrane. Recent studies have suggested that Hfq plays a role in the regulation of DNA replication, mutagenesis, and recombination. In this narrative review, we will focus on the importance of membranes in DNA transactions and discuss the potential role of Hfq-mediated regulation of these processes in Escherichia coli, where the protein is the best characterized. Special attention is given to the affinity of this small protein for both DNA and membranes, which might help explain some of the findings from recent experiments.
    Keywords:  DNA polymerase; Hfq protein; biological membranes; cytoskeletal protein; membrane transertion; outer membrane vesicle cargo; plasmids; transposons
    DOI:  https://doi.org/10.3390/membranes15040103
  5. iScience. 2025 May 16. 28(5): 112292
    Preparation for mitosis requires gradual CDK1 activation.
    Karen Akopyan, Zhiyu Hao, Arne Lindqvist.
      G2 phase is considered as a time in which cells prepare for the large structural changes in the following mitosis. Starting at completion of DNA replication, CDK1 and PLK1 kinase activities gradually increase throughout G2 phase until reaching levels that initiate mitosis. Here, we use a combination of experiments and a data-driven mathematical model to study the connection between DNA replication and mitosis. We find that gradual activation of mitotic kinases ensures CDK1-dependent transcription of factors required for mitosis. In addition, we find that gradual activation of CDK1 coordinates CDK1 and PLK1 activation. Conversely, shortening G2 phase by WEE1 inhibition leads to mitotic delays, which can be partially rescued by expression of constitutively active PLK1. Our results show a function for slow mitotic kinase activation through G2 phase and suggest a mechanism for how the timing of mitotic entry is linked to preparation for mitosis.
    Keywords:  Cell biology; Functional aspects of cell biology; Mathematical biosciences
    DOI:  https://doi.org/10.1016/j.isci.2025.112292
  6. Dev Cell. 2025 Apr 16. pii: S1534-5807(25)00179-0. [Epub ahead of print]
    RIF1 controls replication timing in early mouse embryos independently of lamina-associated nuclear organization.
    Tsunetoshi Nakatani, Tamas Schauer, Mrinmoy Pal, Andreas Ettinger, Luis Altamirano-Pacheco, Julia Zorn, David M Gilbert, Maria-Elena Torres-Padilla.
      Cells must duplicate their genome before they divide to ensure equal transmission of genetic information. The genome is replicated with a defined temporal order, replication timing (RT), which is cell-type specific and linked to 3D-genome organization. During mammalian development, RT is initially not well defined and becomes progressively consolidated from the 4-cell stage. However, the molecular regulators are unknown. Here, by combining loss-of-function analysis with genome-wide investigation of RT in mouse embryos, we identify Rap1 interacting factor 1 (RIF1) as a regulator of the progressive consolidation of RT. Embryos without RIF1 show DNA replication features of an early, more totipotent state. RIF1 regulates the progressive stratification of RT values and its depletion leads to global RT changes and a more heterogeneous RT program. Developmental RT changes are disentangled from changes in transcription and nuclear organization, specifically nuclear lamina association. Our work provides molecular understanding of replication and genome organization at the beginning of mammalian development.
    Keywords:  RIF1; early mouse embryos; lamina-associated domains; replication fork speed; replication timing; single-cell Repli-seq
    DOI:  https://doi.org/10.1016/j.devcel.2025.03.016
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