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1. Replication forks associated with long nuclear actin filaments in mild stress conditions display increased dynamics

   Merigliano, Chiara; Palumbieri, Maria Dilia; Lopes, Massimo; Chiolo, Irene (November 2024, microPublication biology)

   Nuclear actin filaments (F-actin) form during S-phase and in response to replication stress to promote fork remodeling and repair. In mild replication stress conditions, nuclear actin polymerization is required to limit PrimPol recruitment to the fork while promoting fork reversal. Both short and long filaments form during this response, but their function in the nuclear dynamics of replication sites was unclear. Here, we show that replication centers associated with long nuclear actin filaments become more mobile than the rest of the forks, suggesting relocalization of replication sites as a response to prolonged fork stalling and/or fork breakage, even in response to mild replication stress. 

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2. Nuclear actin polymerization rapidly mediates replication fork remodeling upon stress by limiting PrimPol activity

   https://doi.org/10.1038/s41467-023-43183-5  

   Palumbieri, Maria Dilia; Merigliano, Chiara; González-Acosta, Daniel; Kuster, Danina; Krietsch, Jana; Stoy, Henriette; von Känel, Thomas; Ulferts, Svenja; Welter, Bettina; Frey, Joël; et al (December 2023, Nature Communications)

   Abstract Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase and observed their rapid extension in number and length upon genotoxic treatments, frequently taking contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork remodeling is linked to deregulated chromatin loading of PrimPol, which promotes unrestrained and discontinuous DNA synthesis and limits the recruitment of RAD51 and SMARCAL1 to nascent DNA. Moreover, defective nuclear actin polymerization upon mild replication interference induces chromosomal instability in a PRIMPOL-dependent manner. Hence, by limiting PrimPol activity, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments. 

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3. Live Cell Imaging of Nuclear Actin Filaments and Heterochromatic Repair foci in Drosophila and Mouse Cells

   https://doi.org/10.7287/peerj.preprints.27900v1  

   See, C; Arya, D; Lin, E; Chiolo, I (July 2021, Methods in molecular biology)

   null (Ed.)

   Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here, we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair. 

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4. Race against Time between the Virus and Host: Actin-Assisted Rapid Biogenesis of Replication Organelles is Used by TBSV to Limit the Recruitment of Cellular Restriction Factors

   https://doi.org/10.1128/jvi.00168-21  

   Molho, Melissa; Zhu, Shifeng; Nagy, Peter D. (June 2022, Journal of Virology)

   Simon, Anne E. (Ed.)

   ABSTRACT Positive-strand RNA viruses build large viral replication organelles (VROs) with the help of coopted host factors. Previous works on tomato bushy stunt virus (TBSV) showed that the p33 replication protein subverts the actin cytoskeleton by sequestering the actin depolymerization factor, cofilin, to reduce actin filament disassembly and stabilize the actin filaments. Then, TBSV utilizes the stable actin filaments as “trafficking highways” to deliver proviral host factors into the protective VROs. In this work, we show that the cellular intrinsic restriction factors (CIRFs) also use the actin network to reach VROs and inhibit viral replication. Disruption of the actin filaments by expression of the Legionella RavK protease inhibited the recruitment of plant CIRFs, including the CypA-like Roc1 and Roc2 cyclophilins, and the antiviral DDX17-like RH30 DEAD box helicase into VROs. Conversely, temperature-sensitive actin and cofilin mutant yeasts with stabilized actin filaments reduced the levels of copurified CIRFs, including cyclophilins Cpr1, CypA, Cyp40-like Cpr7, cochaperones Sgt2, the Hop-like Sti1, and the RH30 helicase in viral replicase preparations. Dependence of the recruitment of both proviral and antiviral host factors into VROs on the actin network suggests that there is a race going on between TBSV and its host to exploit the actin network and ultimately to gain the upper hand during infection. We propose that, in the highly susceptible plants, tombusviruses efficiently subvert the actin network for rapid delivery of proviral host factors into VROs and ultimately overcome host restriction factors via winning the recruitment race and overwhelming cellular defenses. IMPORTANCE Replication of positive-strand RNA viruses is affected by the recruitment of host components, which provide either proviral or antiviral functions during virus invasion of infected cells. The delivery of these host factors into the viral replication organelles (VROs), which represent the sites of viral RNA replication, depends on the cellular actin network. Using TBSV, we uncover a race between the virus and its host with the actin network as the central player. We find that in susceptible plants, tombusviruses exploit the actin network for rapid delivery of proviral host factors into VROs and ultimately overcome host restriction factors. In summary, this work demonstrates that the actin network plays a major role in determining the outcome of viral infections in plants. 

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5. The role of fork stalling and DNA structures in causing chromosome fragility

   https://doi.org/10.1002/gcc.22721  

   Kaushal, Simran; Freudenreich, Catherine H. (January 2019, Genes, Chromosomes and Cancer)

   Abstract Alternative non‐B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single‐stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT‐rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance. 

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