Temporal coordination between chromosome mobility and homologous recombination

  1. Fraulin Joseph
  2. So Jung Lee
  3. Eric Edward Bryant
  4. Robert J. D. Reid
  5. Ivana Sunjevaric
  6. Rodney Rothstein

  • Curated by eLife

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    Evaluation Summary:

    Damaged chromatin displays an increase in nuclear mobility, but the importance of this response in homologous recombination (HR) repair is under debate. This study shows tight temporal coordination between HR repair events in budding yeast, where the increase in the mobility of repair sites follows resection and precedes chromosome pairing and gene conversion. With several elegant assays, the authors demonstrate that this temporal correlation remains intact in conditions that either delay resection or promote resection. This is consistent with the role of increased mobility in promoting chromosome pairing and HR progression, downstream from resection.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Homologous recombination (HR), a principal cellular pathway for double-strand break (DSB) repair, is linked to changes in chromosome movement. Although increased chromosome mobility in response to a DSB has been observed in a variety of species, its precise role in HR remains controversial. Here, we find that end resection, the recruitment of recombination proteins, increased chromosome mobility, the pairing of homologs and gene conversion are temporally linked in response to a DSB. In mre11Δ mutant cells, which exhibit a delay in the initial processing of a DSB, chromosome mobility and all subsequent recombination events are also delayed. Overexpression of the Dna2 nuclease suppresses the mre11Δ delay in end resection and restores the original timing of chromosome mobility and all subsequent downstream HR events. Thus, changing the timing of chromosome mobility results in a corresponding change in essential downstream HR events, reinforcing its mechanistic role in the DNA repair process.

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  1. eLife

    Evaluation Summary:

    Damaged chromatin displays an increase in nuclear mobility, but the importance of this response in homologous recombination (HR) repair is under debate. This study shows tight temporal coordination between HR repair events in budding yeast, where the increase in the mobility of repair sites follows resection and precedes chromosome pairing and gene conversion. With several elegant assays, the authors demonstrate that this temporal correlation remains intact in conditions that either delay resection or promote resection. This is consistent with the role of increased mobility in promoting chromosome pairing and HR progression, downstream from resection.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  2. eLife

    Reviewer #3 (Public Review):

    When a genome suffers toxic DNA damage such as double-strand breaks (DSBs), the cell sets up detection and repair systems to ensure cell survival. In the last decade, the mobility of both the damaged chromatin and the rest of the genome has emerged as a means of signaling the damage. However, the role of this mobility in the repair process between the damaged sequence and a homologous copy by homologous recombination (HR) remains poorly understood. The authors question the order and the relationship between homologous repair events, namely resection at the break site, recruitment of the recombination machinery, chromosome mobility, and the encounter between damaged sequence and homologous copy (pairing) in order to determine the impact that chromosome mobility may have in HR.

    The strength of this study is that it uses a eukaryote model, budding yeast, where the genetics of DSB repair is extremely well known and where chromosome mobility has been discovered (among others by the team). Yeast cells are naturally diploid or haploid, this study uses diploids allowing for homologous recombination under physiological conditions. In addition, this study tracks DSB repair events over a short time frame, allowing a closer look at the step at which chromosome mobility occurs. The study of resection at the DSB as a regulator of chromosome mobility is thus quite appropriate.

    In order to strengthen the conclusions of this paper, we suggest adding some controls and details to allow adequate matching of results to conclusions. In particular, to be able to control for the delay in the mobility of the ∆mre11 mutant at 4h, the mobility of the wild-type strain at the same time is essential. Similarly, if resection induces an increase in mobility that favors pairing, after pairing, mobility should return to normal: it would be good to show this.
    The use of a haploid strain is interesting because it has a slower resection. Analysis of mobility in the haploid strain would probably confirm the relationship between resection and mobility.

    The community already uses the methods described here. The impact of this study will be to advance the understanding of the regulation of chromosome mobility when DSB happen, an important and innovative parameter for the repair of damage.

  3. eLife

    Reviewer #2 (Public Review):

    In "temporal coordination between chromosomal mobility and homologous recombination", Joseph and colleagues investigated the timing of repair events after inducing a single Double-Strand Break (DSB) in yeast diploid cells. In particular, the authors focus on the events of resection, chromatin mobility, homologue chromosomes pairing, and repair by gene conversion, and demonstrated how a delay in the early event of resection due to the deletion of the MRX component MRE11 corresponds to a delay in all the other steps. Interestingly, they also showed that, when the proper timing of resection is restored in mre11D cells by the overexpression of DNA2, also the timing of DSB mobility, pairing, and repair is restored.

    The temporal correlation between DSB induction, DNA resection, chromosome pairing, and repair is well supported by the data. In addition, data showing the delay in processing, mobility, and repair in mre11D cells are clear and well presented, and the suppression by DNA2 overexpression is also well characterized. However, the author's claim that chromatin mobility is required for DSBs repair by promoting pairing is not completely demonstrated.

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, the authors build on prior work from their group to investigate the mechanisms of DNA double-strand break (DSB) mobility and its consequences for homology-directed repair in budding yeast. The authors observe that the kinetics of the initial processing of the DSB (end resection, modeled by cells lacking Mre11) predicts the timing of subsequent events, including the DSB-induced increase in mobility, loading of RPA and formation of the nucleoprotein filament, and homolog pairing leading to gene conversion (GC) in this diploid model system. Over-expression of the nuclease Dna2 but not Exo1 rescues all molecular defects tied to resection and GC arising from loss of Mre11 in a manner that surprisingly is not reflected in cell growth after irradiation. Taken together, the authors argue that these data reinforce the notion that increased mobility of DSB downstream of resection defines the kinetics of GC, a hypothesis that has remained somewhat in question.

    In general, the data are of high quality, the data are clearly presented (including useful cartoons in most figures) and the manuscript is well-written. The data support the authors' conclusions. Some challenges in dissecting the causality of increased DSB mobility remain as tools to separate the function of factors that influence DSB mobility versus those that are required for other events in homology search (e.g. nucleosome remodeling, Rad51 loading) are still difficult to untangle, even despite the efforts in this study. The impact that this work will have on the field is somewhat challenging to assess as most of the observations recapitulate those made previously for other resection modulators or repair factors including Rad51 (by this group) and Sae2 (also investigated by the Longhese group). Based on this, one might conclude that the requirement for resection in driving enhanced mobility of the DSB is well established. However, as the authors point out, there has been some evidence put forward that seemingly contradicts this model. A more detailed discussion of the differences in the experimental details employed by this current and prior studies is warranted and would help the reader and field weigh the evidence.

  5. Version published to 10.1101/2022.03.24.485580 on bioRxiv