Budding yeast complete DNA synthesis after chromosome segregation begins

Ivanova, Tsvetomira; Maier, Michael; Missarova, Alsu; Ziegler-Birling, Céline; Dam, Monica; Gomar-Alba, Mercè; Carey, Lucas B.; Mendoza, Manuel

doi:10.1038/s41467-020-16100-3

Cited by 39 publications

(30 citation statements)

References 55 publications

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“…Mitotic DNA repair synthesis (MiDAS) has been documented as a means to complete replication of hard-to-duplicate genome features such as common fragile sites and telomeres ( Özer and Hickson, 2018 ). Moreover, in the unperturbed cell cycles of both yeast ( Ivanova et al, 2020 ; Torres-Rosell et al, 2007 ) and mammalian cells ( Moreno et al, 2016 ) onset of mitosis has been documented in the presence of under-replicated genomic regions, leading to DNA synthesis in offspring cells. These findings suggest that a temporal separation between DNA replication and chromosome segregation does not seem to be a strict rule for eukaryotic cells.…”

Section: Discussionmentioning

confidence: 99%

“…Despite segregation of under-replicated chromosomal regions being mutagenic ( Durkin and Glover, 2007 ; Debatisse et al, 2012 ; Song et al, 2014 ), increased cell division rate in these circumstances may compensate for loss of fitness due to mutations. Also, if segregation of under-replicated loci is limited to specific genome compartments, such as the telomeres and subtelomeres, these may serve as a genetic playground, with higher mutation and copy-number variation allowing for increased genetic diversity, as has been proposed in yeast ( Ivanova et al, 2020 ; Brown et al, 2010 ). Consistent with this idea, our data suggest that chromosome subtelomeric DNA replication is mainly detectable at the temporal extremes of the conventional Leishmania S phase (either very early in S phase, as seen with the G1 enriched population; or very late in S phase, as seen with the G2/M enriched populations), and such regions have been demonstrated to be particularly prone to copy- number variation ( Bussotti et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

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Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

Damasceno

Marques

Beraldi

et al. 2020

eLife

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DNA replication is needed to duplicate a cell's genome in S-phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S-phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor colocalise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1 phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania's genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S-phase, to support predominantly chromosome-internal DNA replication initiation within S-phase.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

Damasceno

Marques

Beraldi

et al. 2020

eLife

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“…The different properties of Cdc28 variants found in our study advocates for the existence of several ways of suppression of pol2rc-ΔN defect. One explanation is based on the observation that the proper level of Cdc28 is required to complete the cell cycle when replication extends beyond chromosome segregation [79]. In the pol2rc-ΔN cells, the proportion of under-replicated genomic regions might be higher than in normal cells, as indicated by aberrant cell and nucleus morphology (Fig.…”

Section: Discussionmentioning

confidence: 99%

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations inCDC28gene

Stepchenkova

Zhuk

Cui

et al. 2020

Preprint

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DNA polymerase ε (pol ε) participates in the leading DNA strand synthesis in eukaryotes. The catalytic subunit of this enzyme, Pol2, is a fusion of two ancestral B-family DNA polymerases. Paradoxically, the catalytically active N-terminal pol is dispensable, and an inactive C-terminal pol is essential for yeast cell viability. Despite extensive studies of strains without the active N-terminal part (mutation pol2-16), it is still unclear how they survive and what the mechanism is of rapid recovery of initially miserably growing cells. Slow progress is attributed to the difficultly of obtaining strains with the defect. We designed a robust method for the construction of mutants with only the C-terminal part of Pol2 using allele pol2rc-ΔN optimized for high protein production. Colonies bearing pol2rc-ΔN appear three times sooner than colonies of pol2-16 but exhibit similar growth defects: sensitivity to hydroxyurea, chromosomal instability, and an elevated level of spontaneous mutagenesis. UV-induced mutagenesis is partially affected, it is lower only at high doses in some reporters. The analysis of the genomes of pol2rc-ΔN isolates revealed the prevalence of nonsynonymous mutations suggesting that the growth recovery was a result of evolution by positive selection for better growth fueled by variants produced by the elevated mutation rate. Mutations in the CDC28 gene, the primary regulator of the cell cycle, were repeatedly found in independent clones. Genetic analysis established that cdc28 alleles single-handedly improve the growth of pol2rc-ΔN strains and suppress HU-sensitivity. The affected amino acids are located on the Cdc28 molecule’s surfaces that mediate contacts with cyclins and kinase subunits. Our work establishes the significance of the CDC28 gene for the resilience of replication and predicts that changes in mammalian homologs, cyclin-dependent kinases may play a role in remastering replication to compensate for the defects in the leading strand synthesis by the dedicated polymerase.Author SummaryThe catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable while the inactive C-terminal part is required for viability. The corresponding strains show a severe growth defect, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly produced fast-growing clones. We discovered that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs during evolution by positive selection for a better growth fueled by variants produced by elevated mutation rates. Mutations in the cell cycle-dependent kinase gene, CDC28, can single-handedly improve the growth of strains lacking the N-terminal part of Pol2. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may play a role in response to the defects of active leading strand polymerase.

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“…These regions would include centromeres, G-quadruplex, fragile sites, transposable elements, non-coding RNAs and subtelomeric regions [18,54,[59][60][61]. Very recently, another paper has shown that pericentromeric regions accumulate DNA damage markers during S-phase [62].…”

Section: Discussionmentioning

confidence: 99%

Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes

Ayra-Plasencia

Ramos-Pérez

Santana-Sosa

et al. 2020

Preprint

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The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and even anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses and GFP-tagged DNA damage markers suggest that non-recombinational modifications of late replication intermediates may account for the shift in the PFGE behavior. The fact that this shift does not trigger G2/M checkpoints further supports this statement since checkpoints are active for other replicative stresses in the absence of Top2. We propose that the prolonged absence of Top2 activity leads to a general chromosome structural change. This change might interlock chromatids together with catenations and thus contribute to the formation of anaphase bridges in top2 mutants.

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Budding yeast complete DNA synthesis after chromosome segregation begins

Cited by 39 publications

References 55 publications

Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations inCDC28gene

Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes

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