bims-replis 2025-07-27 papers

J Bacteriol. 2025 Jul 21. e0017525

DNA replication initiation timing is important for maintaining genome integrity.

Tristan T Reed, Abigail H Kendal, Katherine J Wozniak, Lyle A Simmons.

DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of replication initiation, while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of replication on genome stability remain unclear. In this work, we measure the origin-to-terminus ratio as a proxy for replication initiation activity. We show that ΔccrZ and several ccrZ alleles under-initiate DNA replication, while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by the low frequency formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA-damaging agents. We show that cells under-initiating and over-initiating DNA replication are both sensitive to mitomycin C, demonstrating that changes in replication initiation frequency cause an increase in sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to asynchronous DNA replication, leading to inefficient homologous recombination. In cells over-initiating replication, we propose that an increase in the number of replication forks leads to replication fork stress, which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled because changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.IMPORTANCEThe regulation of DNA replication is fundamental to cell growth and cell cycle control. In eukaryotes, under-initiation or over-initiation leads to genome instability. In bacteria, it is unclear how changes in replication initiation frequency impact DNA replication status and genome integrity. We show that tight regulation of DNA replication initiation is critical for maintaining genome integrity. Cells over-initiating or under-initiating DNA replication are sensitive to DNA damage. Furthermore, cells over-initiating DNA replication experience replication fork stress at levels that phenocopy those observed in cells challenged with DNA damage from mitomycin C. Our results establish the critical importance of properly regulating DNA replication initiation frequency because an imbalance in initiation results in replication fork perturbations, deficiencies in DNA repair, and genome instability.

Keywords: Bacillus subtilis; CcrZ; DNA replication; DnaA; RecA

DOI: https://doi.org/10.1128/jb.00175-25

Nucleic Acids Res. 2025 Jul 19. pii: gkaf679. [Epub ahead of print]53(14):

Evidence that transient replication errors initiate nuclear genome mutations.

Scott A Lujan, Zhi-Xiong Zhou, Thomas A Kunkel.

DNA synthesis during genomic replication generates mismatches that lead to mutations. Point mutations may be caused by base-base mismatches that yields base substitutions or by primer- or template-strand slippage, which yield insertions and deletions (indels), respectively. Evidence obtained 40 years ago with DNA polymerases in vitro indicated that transient DNA intermediates also initiate substitutions and indels. Here, we provide evidence in vivo that the rates of specific single-base mutations at or adjacent to the 3'-terminus of the primer strand of mononucleotide runs increase change with run length. We propose that four such TIM (transient initiator mutagenesis) pathways are active during replication of the yeast nuclear genome in vivo and may be a universal feature of DNA replication.

DOI: https://doi.org/10.1093/nar/gkaf679

Nat Commun. 2025 Jul 21. 16(1): 6685

Mechanism of Rad51 filament formation by Rad52 and Rad55-Rad57 in homologous recombination.

Jaigeeth Deveryshetty, Ayush Mistry, Sushil Pangeni, Mohamed Ghoneim, Monica Tokmina-Lukaszewska, Steven K Gore, Jie Liu, Vikas Kaushik, Simrithaa Karunakaran, Angela Taddei, Wolf-Dietrich Heyer, Taekjip Ha, Brian Bothner, Edwin Antony.

Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs) by generating single-stranded DNA (ssDNA), which is initially coated by Replication Protein A (Rpa). Rad51, a recombinase, catalyzes strand invasion but binds ssDNA with lower affinity than Rpa, necessitating mediator proteins like Rad52 (yeast) or BRCA2 (humans) for Rad51 loading. The mechanisms of this exchange remain unclear. We show that Saccharomyces cerevisiae Rad52 uses its disordered C-terminus to sort polydisperse Rad51 into discrete monomers. Using fluorescent-Rad51 and single-molecule optical tweezers, we visualize Rad52-mediated Rad51 filament formation on Rpa-coated ssDNA, preferentially at ssDNA-dsDNA junctions. Deleting the C-terminus of Rad52 disrupts Rad51 sorting and loading. Addition of the Rad51 paralog Rad55-Rad57 enhances Rad51 binding by ~60%. Despite structural differences, Rad52 and BRCA2 share conserved functional features. We propose a unified "Sort, Stack & Extend" (SSE) mechanism by which mediator proteins and paralogs coordinate Rad51 filament assembly during HR.

DOI: https://doi.org/10.1038/s41467-025-61811-0

Curr Oncol. 2025 Jun 25. pii: 370. [Epub ahead of print]32(7):

Unveiling ctDNA Response: Immune Checkpoint Blockade Therapy in a Patient with POLE Mutation-Associated Early-Onset Colon Cancer.

Ramya Ramachandran, Marisa Cannon, Supriya Peshin, Madappa Kundranda, Aaron J Scott.

Colorectal cancer (CRC) is the third most common malignancy worldwide and the second leading cause of cancer-related mortality in the United States. The incidence of early-onset colorectal cancer (EOCRC) has been increasing over the past several decades. While the etiologies for this rising incidence remain unclear, genetic factors likely play an important role. DNA polymerase epsilon (POLE) mutations occur at a higher rate than average-onset colorectal cancer (AOCRC). DNA polymerase epsilon (Pol ε) is a high-fidelity, processive polymerase that is a promising target for immune checkpoint inhibitors due to its association with various human malignancies, including colorectal cancer. EOCRC remains a major area of focus, and POLE mutations leading to the high-TMB subtype constitute a potential therapeutic target.

Keywords: Ipilimumab; Nivolumab; POLE; case report; ctDNA; early onset colorectal cancer

DOI: https://doi.org/10.3390/curroncol32070370

J Biomol NMR. 2025 Jul 22.

Divide-and-conquer strategy for NMR studies of the E. coli γ-clamp loader complex.

Sam Mahdi, Irina V Semenova, Irina Bezsonova, Penny J Beuning, Dmitry M Korzhnev.

The E. coli γ-clamp loader is a 200 kDa pentameric AAA + ATPase comprised of γ, δ and δ' subunits in a 3:1:1 ratio, which opens the ring shaped β-clamp homodimer and loads it onto DNA in a process essential for DNA replication. The clamp loading is initiated by ATP binding, which induces conformational changes in the clamp loader allowing it to bind and open the β-clamp. This is followed by DNA primer-template binding, ATP hydrolysis, and clamp release onto DNA. Despite a wealth of structural and functional data, dynamics and interactions of the γ-clamp loader and the β-clamp underlying elementary steps of this process remain elusive. Here we employed a "divide-and-conquer" strategy for the initial NMR characterization of the γ-clamp loader. A new protocol for the clamp loader assembly was proposed allowing selective incorporation of the isotope-labeled δ and δ' subunits for NMR studies. The nearly complete 1H, 15N and 13C NMR resonance assignments were obtained for the isolated modular domains of the δ and δ' subunits, which facilitated the assignments of the full-length subunits, and side-chain methyl assignments of the subunits in the context of pentameric γ-clamp loader. NMR chemical shift analysis using the random coil index approach revealed increased flexibility in the ATP, DNA, and β-clamp binding interfaces of the isolated subunits, highlighting a potential significance of conformational dynamics for the clamp loading process. The reported clamp loader assembly protocol and resonance assignments enable the detailed NMR studies of protein dynamics and mechanochemistry of the clamp loading cycle.

Keywords: DNA replication; DnaX; HolA; HolB; TROSY NMR; β processivity clamp

DOI: https://doi.org/10.1007/s10858-025-00471-0