Budding yeast complete DNA replication after chromosome segregation begins

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).

 

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 disassembly^4,5 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: 25 September 2018 , updated on: 11 May 2020

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

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