Menu

Close

Budding yeast complete DNA replication after chromosome segregation begins

Tsvetomira Ivanova, Michael Maier, Alsu Missarova, Celine Ziegler-Birling, Lucas B. Carey, Manuel Mendoza

Preprint posted on September 05, 2018 https://www.biorxiv.org/content/early/2018/09/05/407957

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-020-16100-3

Procrastinating polymerases: Budding yeast postpone the replication of tricky DNA sequences until anaphase

Selected by Dey Lab, Maiko Kitaoka

Categories: biochemistry, cell biology

Note added 11th May 2020: This preprint now published as “Budding yeast complete DNA synthesis after chromosome segregation begins” at Nature Communications. 

[This post was co-authored with Delyan Mutavchiev at the MRC LMCB, UCL, London]

Context

The life cycle of every dividing cell must include the faithful duplication and segregation of its genetic material. While in prokaryotes these two processes can occur simultaneously1, eukaryotes are thought to regulate this timing more carefully. Cells that delay replication or initiate chromosome segregation prematurely accumulate a range of genetic defects in a phenomenon referred to as genome instability2. DNA replication in S phase is temporally isolated from mitotic onset and subsequent chromosome segregation by the G2 gap phase. The sequential ordering of cell cycle phases is under the master control of cyclin-dependent kinase (Cdk) activity. In response to major replication or segregation defects, cell cycle checkpoints enforce temporal ordering by arresting cells until the underlying defects are corrected3.

However, it is unclear if DNA replication completion is strictly required to initiate mitotic entry. In fact, Ivanova, Maier, Missarova et al. demonstrate intriguing evidence of the opposite in budding yeast. They show that during unperturbed growth, DNA replication is completed after the onset of chromosome segregation, without bypassing control by Cdk activity (Fig 1).

Figure 1: adapted from Figure 4D of Ivanova, Meier, Missarova et al. 2018 with the authors’ permission under a CC-BY 4.0 international license. Figure highlights a second phase of replication (“S2”) after Cdk levels drop in anaphase.

 

Key findings

The authors use EdU incorporation to demonstrate that DNA replication can occur between metaphase and the G1 phase of the next cell cycle. In fact, this mitotic DNA synthesis promotes timely chromosome segregation since disruption of DNA replication in late mitosis causes a delay in nuclear and cell division, as well as the formation of chromatin bridges. Mutants carrying defects in Mitotic Exit Network (MEN) proteins, which have high levels of mitotic cyclins in late anaphase, show increased numbers of chromatin bridges. Their resolution is dependent on the function of the replication machinery, but not on the activity of Topoisomerase II.

 Notably, the authors used Illumina sequencing to measure DNA copy number at various time points in the cell cycle to determine which specific genomic regions were replicated in mitosis. Genomic sequences replicated during late mitosis are mostly found in sub-telomeric regions as well as difficult-to-replicate regions such as G-quadruplexes, fragile sites, and transposable elements.

The authors also present evidence that mitotic replication is suppressed by high Cdk activity in metaphase and permitted during anaphase when Cdk activity is low, since inactivation of Cdk during metaphase arrest allows DNA replication to occur. Inactivation of Cdk in MEN mutants also allowed for the resolution of chromatin bridges.

Tying the sequencing data to their Cdk experiments, the authors suggest that replication forks are likely to stall at difficult-to-replicate regions, but these cannot be replicated in metaphase while high Cdk activity is inhibitory. Replication can only be completed late in mitosis once Cdk levels have decreased. Additionally, genes in mitotic replicating regions have higher intra-species diversity. Noting the paucity of protein-coding genes in these regions, the authors argue that potentially error-prone mitotic replication could actually be exploited as a source of increased genetic diversity.

 

Questions for the authors (and potential future directions?)

 If budding yeast cells are forced to grow slowly (i.e. in minimal medium), would they be more likely to complete replication before mitosis?

Does mitotic replication occur in other fast-growing systems – in particular, those with a relatively long G2- fission yeast, mammalian stem cells or during early stages of embryogenesis?

Anaphase only lasts for a few minutes. Is there something different about late mitotic chromosomes that permits replication and how is the replication process affected by the condensed state of the chromosomes?

If replication occurs in late stages of mitosis, how does some of the replication machinery bypass disassembly4,5 at the end of S-phase or at the start of mitosis?

How conserved is this phenomenon, especially given that budding yeast seem to lack a checkpoint to determine if DNA replication is complete?

 

References:

  1. Jensen, R. B. Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus. J. Bacteriol. 188, 2244–53 (2006).
  2. Aguilera, A. & García-Muse, T. Causes of Genome Instability. Annu. Rev. Genet. 47, 1–32 (2013).
  3. Shaltiel, I. A., Krenning, L., Bruinsma, W. & Medema, R. H. The same, only different – DNA damage checkpoints and their reversal throughout the cell cycle. J. Cell Sci. 128, 607–20 (2015).
  4. Maric, M., Maculins, T., De Piccoli, G. & Labib, K. Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication. Science (80-. ). 346, 1253596 (2014).
  5. Sonneville, R. et al. CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis. Nat. Cell Biol. 19, 468–479 (2017).

 

Tags: cell cycle, checkpoints, s phase

Posted on: 25th September 2018 , updated on: 11th May 2020

doi: https://doi.org/10.1242/prelights.4924

Read preprint (No Ratings Yet)




The authors respond to questions raised in the post

Manuel Mendoza shared

1. If budding yeast cells are forced to grow slowly (i.e. in minimal medium), would they be more likely to complete replication before mitosis?

A. We observed mitotic DNA synthesis in both complete and minimal medium. However, we did not test if slowing the rate of cell division even further would allow replication to finish before mitosis. Perhaps related to this, and as one of the reviewers pointed out, there is some evidence that generally slowing the cell cycle stabilizes the genome [1].

2. Does mitotic replication occur in other fast-growing systems – in particular, those with a relatively long G2- fission yeast, mammalian stem cells or during early stages of embryogenesis?

A. We don’t yet know if anaphase DNA synthesis occurs in other systems, fast-growing or not. However, the idea that yeast cells complete DNA replication in anaphase has interesting parallels with mitotic DNA synthesis (MiDAS), a phenomenon observed in mammalian cells exposed to replication stress [2,3]. In the peer-reviewed version of our paper, we investigated this issue further. We find that like MiDAS, yeast anaphase DNA synthesis depends on the DNA polymerase delta subunit Pol32, and does not require Rad51, opening the possibility that both processes are mechanistically related. Another possible similarity between human MiDAS and yeast anaphase DNA synthesis is that both may be inhibited by high CDK levels, since MiDAS was not detected in nocodazole-arrested cells (in which M-Cdk levels are highest) whereas it has been observed in prophase, when M-Cdk levels are relatively lower [2]. It is not known whether MiDAS-like processes occur in anaphase in unstressed mammalian cells. We speculate that DNA synthesis during late mitosis could contribute to resolving ultrafine chromatin bridges, whose defective processing is associated with genome instability.

3. Anaphase only lasts for a few minutes. Is there something different about late mitotic chromosomes that permits replication and how is the replication process affected by the condensed state of the chromosomes?

A. First, it is good to remember that yeast chromosomes are most condensed during anaphase, and therefore decondensation is not necessarily associated with anaphase DNA synthesis. Nevertheless, one could speculate that there is something about the chromatin state in anaphase that favours replication of “tricky” DNA sequences. But we simply do not know. At some point we thought that chromatin stretching due to anaphase spindle elongation could be a factor. However, inhibition of CDK in cells with short spindles (after Cdc20 depletion) is sufficient for DNA copy number to double in subtelomeric regions – so spindle forces are unlikely to be a strong driver of DNA synthesis in these regions.

4. If replication occurs in late stages of mitosis, how does some of the replication machinery bypass disassembly at the end of S-phase or at the start of mitosis?

A. Again, we do not know. It’s worth noting that we don’t even know if DNA synthesis in anaphase is due to replisomes assembled in S phase, or to a different process. This relates to one of the main conceptual changes in our manuscript. Reviewers correctly pointed out that since the mechanism(s) of DNA synthesis in late mitosis is not clear yet, the term “DNA replication” in the title and abstract of our submitted manuscript could be misleading. Readers not in the field of DNA replication might think that “normal” DNA replication is continuing during chromosome segregation. Although that is our preferred hypothesis, our data do not actually demonstrate it. For this reason, we used the more general term “DNA synthesis”.

5. How conserved is this phenomenon, especially given that budding yeast seem to lack a checkpoint to determine if DNA replication is complete?

A. See our reply to question 2, above. Perhaps anaphase DNA synthesis such as “anaphase MiDAS” does occur in some animal cells and it has not been detected yet. We hope our study will inspire further research in this direction.

1. Vinton, P. J. & Weinert, T. A Slowed Cell Cycle Stabilizes the Budding Yeast Genome. Genetics 206, 811–828 (2017).

2. Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Dey Lab

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Preprint missing? Don't hesitate to let us know.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close