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Actin nucleators safeguard replication forks by limiting nascent strand degradation

Jadwiga Nieminuszczy, Peter R. Martin, Ronan Broderick, Joanna Krwawicz, Alexandra Kanellou, Camelia Mocanu, Vicky Bousgouni, Charlotte Smith, Kuo-Kuang Wen, Beth L. Woodward, Chris Bakal, Fiona Shackley, Andres Aguilera, Grant S. Stewart, Yatin M. Vyas, Wojciech Niedzwiedz

Posted on: 22 March 2023 , updated on: 23 March 2023

Preprint posted on 12 January 2023

Article now published in Nucleic Acids Research at http://dx.doi.org/10.1093/nar/gkad369

Actin’ in the nucleus: better safe than broken

Selected by Roberto Amadio

Background

DNA replication precedes cell division. Duplication of genetic information within a cell must be as much accurate as possible, to prevent propagation of mutations and potential onset of diseases. During replication, DNA is unwound by the replication fork to generate two single stranded DNA molecules (ssDNA) serving as templates for replication. Replication Protein A (RPA) is recruited to ssDNA filaments to protect them and resolve replicational stress that may arise during duplication. Actin and Actin Nucleator Promoting Factors (NPFs) have been suggested to participate in the DNA damage response and replication, but still the molecular mechanisms underlying their function in these essential cellular processes remain largely unknown. In this preprint, the authors show that multiple NPFs are recruited to replication forks and facilitate the formation of RPA-ssDNA complexes. Moreover, actin monomers were found to directly interact with RPA and assist its functions. In summary, this preprint describes an emerging function for actin cytoskeletal components inside the nucleus and their involvement in genomic stability and DNA replication.

 

Key findings of this preprint

  1. Nuclear NPFs associate with the replication fork

Starting from iPOND (isolation of proteins on nascent DNA) datasets, the authors identified several proteins involved in actin polymerization (i.e. Arp2/3 complex, N-Wasp and Diaph formins). To experimentally confirm their presence on nascent DNA, they used both iPOND-western blot and Proximity Ligation Assays (PLA). Using both methods, WASp, N-WASp, as well as Diaph1 were shown to associate with the replication fork and this association was augmented upon the induction of replication stress, suggesting that these actin-related factors are actively recruited to sites of replication crisis.

 

  1. Blocking actin polymerization by interfering with NPFs’ function leads to replication stress and DNA damage

The authors used the DNA fiber assay to monitor the impact of NPFs on replication stress. This assay involves the sequential incubation of different thymidine analogs that can later be detected by differentially labelled antibodies. By administering a DNA damage agent (in this case Hydroxyurea, HU) alongside the second thymidine analog, it is possible to quantify the length of this second labelled DNA fiber as a measure of DNA replication proficiency. Targeting NPFs’ activity either by specific siRNA, small molecules inhibitors or by using Diaph1-deficient cells, the authors found a reduced length of second labelled DNA fiber, suggesting a defective replicational fork velocity and inefficient restart upon stress induced by HU (Fig. 1).

Fig. 1 – DNA fiber assay in control and cells depleted for the specified NPF.

 

When trying to combine Arp2/3 inhibition with formins inhibition, the authors found an exasperated phenotype, indicating that Arp2/3 and formins use different and complementary pathways to ensure replicational fork stability. Moreover, assessing 53BP1 and micronuclei formation as a proxy to quantify DNA damage, the authors observed an increase in both these parameters at steady state as well as upon stress induction in NPFs-depleted cells, suggesting a role for actin polymerization in genome stability both at steady state and upon replication stress.

 

  1. RPA-ssDNA binding is helped by NPFs

By applying a modified DNA fiber assay (Fig. 2), the author tested the degradation of DNA filaments in wild-type and NPFs-depleted cells. The depletion of NPFs resulted in increased DNA degradation, which was then shown to be dependent on a “chaperoning” role of NPFs exerted on the RPA-ssDNA interaction. Indeed, increasing RPA expression was sufficient to overcome this lacking chaperoning activity and to restore RPA-ssDNA interaction in NPFs depleted cells.

Fig. 2 – Modified DNA fiber assay to monitor ssDNA degradation upon replication stress in control and NPFs depleted cells.

 

  1. Actin binds RPA and provides a second layer of replication fork stabilization

Finally, the authors provided some evidence that actin polymerization by itself may provide a complementary mechanism to ensure proper DNA replication. Globular actin (G-actin) was found to directly interact with RPA and mutations in G-actin that led to defective actin polymerization were found to sufficiently phenocopy the loss of NPFs.

In conclusion, the authors propose a double mechanism of the actin polymerization machinery – comprising both NPFs’ functions as well as the actin polymerization process itself – in preserving and regulating proper DNA replication in cells.

 

Why I chose this preprint

The function of actin and NPFs inside the nuclei of different cell types is an intriguing and relatively new topic of which we still know very little. I personally liked the experiments and the discussion provided by the authors in this preprint. I think it provides important new information to this emerging field of research and stimulates the reader to reflect on parallel between common phenotypes shown by patients suffering from actin polymerization defects and DNA replication and repair defects, hinting at a closer, yet not well-characterized interaction between these two biological aspects.

 

Questions to the authors

  • Does NPFs’ nuclear localization increase upon HU treatment or during replication stress?
  • Does Diaph1 have a clear cell type expression pattern like WASp and N-WASp?
  • Would it be possible to pharmacologically increase RPA function to overcome replication defects in NPFs depleted cells?

 

References

· Zeman, M.K. and Cimprich, K.A. (2014) Causes and consequences of replication stress. Nature cell biology, 16, 2–9

· Caridi, C.P., Plessner, M., Grosse, R. and Chiolo, I. (2019) Nuclear actin filaments in DNA repair dynamics. Nature cell biology, 21, 1068–1077

Tags: actin, dna damage, dna replication, replication fork

doi: https://doi.org/10.1242/prelights.34147

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