Nuclear myosin VI maintains replication fork stability

Jie Shi, Kristine Hauschulte, Ivan Mikicic, Srijana Maharjan, Valerie Arz, Jan B. Heidelberger, Jonas V. Schaefer, Birgit Dreier, Andreas Plückthun, Petra Beli, Helle D. Ulrich, Hans-Peter Wollscheid

Preprint posted on 31 July 2022 https://www.biorxiv.org/content/10.1101/2022.07.28.501567v1

Myosin VI: a new shield to protect replication forks

Selected by Pierre Caron

Categories: cancer biology, cell biology, molecular biology

Background

The duplication of our genome is a challenging time since replication forks encounter many obstacles that could lead to their collapse. Following replication stress, the fork reversal process reverses fork direction to form a Holliday junction-like structure. This protective mechanism ensures proper DNA damage repair and replication restart. However, the fork ends that result from fork reversal are subject to degradation by nucleases which is linked to increased genomic instability. Among the different key players in reversed fork protection, Breast And Ovarian Cancer Susceptibility Protein 1 and 2 (BRCA1 and BRCA2) and The ATPase Werner Helicase-Interacting Protein 1 (WRNIP1) play a central role. Indeed, these factors counteract the nucleolytic activity against fork ends of MRE11/MUS81 and DNA2, SLX4/ERCC1 nucleases, respectively. Similarly, proteins implicated in chromatin dynamics were found to promote reversed fork protection following replication stress (Quinet et al., 2017; Tye et al., 2021).

Considering the emerging roles of nuclear cytoskeleton proteins in regulating DNA repair and DNA replication (Caridi et al., 2019), a role for these factors in regulating reversed fork stability could be anticipated.

In this preprint, the Ulrich group unveils how Myosin VI protects reversed forks from nucleolytic attacks.

Key Findings

In human cells, the authors showed that Myosin VI interacts with DNA repair factors and many replication-associated factors and found that this factor is required for efficient DNA replication. Remarkably, following replication stress induced by hydroxyurea (HU), the absence of nuclear Myosin VI led to reversed fork degradation to the same extent as could be found in conditions where BRCA2 and WRNIP1 were depleted. In addition, Myosin VI directly associated with damaged forks (Fig 1A) and protected them from nucleolytic degradation by DNA2, but not MRE11. Mechanistically, the authors found that Myosin VI is required for WRNIP1 accrual at reversed forks following replication stress (Fig 1B), thus preventing DNA2 activity toward fork ends.

This study thus uncovers that the nuclear cytoskeleton factor Myosin VI is required for reversed fork protection and genomic stability through a pathway involving the factor WRNIP1, but distinct from BRCA2.

Figure 1 with panels from (Jie Shi et al., 2022)

Figure 1 with panels from (Jie Shi et al., 2022).

A. Representative micrographs of in situ protein interactions at a nascent replication forks (SIRF) assay in human U2OS cells to monitor PCNA, WRNIP1 and Myosin VI (Myo6) accrual levels at fork replication following (or not) DNA replication stress induced by hydroxyurea (HU).

B. Representative micrographs of SIRF assay in human U2OS cells either proficient (siCtrl) or depleted for Myosin VI (siMyo6) to monitor PCNA, WRNIP1 levels at replication fork following (or not) DNA replication stress induced by HU.

References

Caridi, C. P., Plessner, M., Grosse, R., & Chiolo, I. (2019). Nuclear actin filaments in DNA repair dynamics. Nature Cell Biology, 21(9), 1068–1077. https://doi.org/10.1038/s41556-019-0379-1

Jie Shi, Kristine Hauschulte, Ivan Mikicic, Srijana Maharjan, Valerie Arz, Jan BHeidelberger, Jonas V. Schaefer, Birgit Dreier, Andreas Plückthun, Petra Beli, Helle D. Ulrich, & Hans-Peter Wollscheid. (2022). Nuclear myosin VI maintains replication fork stability. BioRxiv.

Quinet, A., Lemaçon, D., & Vindigni, A. (2017). Replication Fork Reversal: Players and Guardians. Molecular Cell, 68(5), 830–833. https://doi.org/10.1016/j.molcel.2017.11.022

Tye, S., Ronson, G. E., & Morris, J. R. (2021). A fork in the road: Where homologous recombination and stalled replication fork protection part ways. Seminars in Cell & Developmental Biology, 113, 14–26. https://doi.org/10.1016/j.semcdb.2020.07.004

What I liked about this preprint

I really like this study presented by the Ulrich group as it unveils a new role for nuclear cytoskeleton factors in regulating DNA replication and sheds light on the mechanism that regulates WRNIP1 accrual to fork ends in response to replication stress.

Considering the emerging roles of nuclear cytoskeleton factors in promoting genome stability by regulating DNA repair processes and chromatin mobility in response to DNA damage, this study therefore opens up new perspectives on how genome integrity is regulated by these factors.

Questions to the authors

Q1: Do you think that Myosin VI can protect damaged replication forks by also acting on their position in the nucleus, together with other elements of the nuclear cytoskeleton, in order to avoid aberrant recombination processes between different forks that might be physically close to each other?

Q2: Your study also unveils a crucial role for the Ubiquitin binding domains (MIU and MyUB) of Myosin VI in reversed fork protection. Could you share your thoughts about the interplay between Myosin VI and the ubiquitin system in response to replication stress? Do you envision a role for an ubiquitin ligase in Myosin VI accrual to reversed forks in response to replication stress?

Tags: dna replication, myosin vi

Posted on: 13 December 2022

doi: https://doi.org/10.1242/prelights.33286

Read preprint

(1 votes)

Author's response

Hans-Peter Wollscheid shared

Questions to the authors

A1: Here we can only speculate. Considering the role of F-actin in HDR, where it supports double strand break mobility to facilitate homology search and its reported function in the relocalization of PCNA positive replication stress foci, we can well imagine a role of myosin VI in the (re-) distribution of (a certain pool of) damaged replication forks. It will be very interesting to explore further cytoskeletal elements in their contribution to replication fork stability. From a myosin point of view, there is an additional aspect to consider. Myosin VI is the only described pointed end-directed motor, while the rest of the myosin superfamily moves towards the barbed end of actin filaments. We still do not know whether nuclear actin filaments are arranged with a specific orientation. In that case, it would be interesting to investigate a potential competition between plus and minus end directed motors.

A2: The contribution of myosin VI´s ubiquitin binding domains (UBDs) to fork stability is very intriguing, Although ubiquitylation is known to regulate virtually all cellular processes, very little is known about ubiquitin modification during the process of fork reversal and reversed fork protection. Not only myosin VI but also WRNIP1 harbors ubiquitin binding properties, making it tempting to speculate about a role of this posttranslational modification at least in this particular branch of the pathway. The identification of ubiquitin ligases and their respective targets is one of the future challenges that will help us to characterize fork reversal as a major genome stability pathway in more detail.

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