The circadian clock ensures successful DNA replication in cyanobacteria

Abstract

Disruption of circadian rhythms causes decreased health and fitness, and evidence from multiple organisms links clock disruption to dysregulation of the cell cycle. However, the function of circadian regulation for the essential process of DNA replication remains elusive. Here, we demonstrate that in the cyanobacterium Synechococcus elongatus, a model organism with the simplest known circadian oscillator, the clock generates rhythms in DNA replication to minimize the number of open replication forks near dusk that would have to complete after sunset. Metabolic rhythms generated by the clock ensure that resources are available early at night to support any remaining replication forks. Combining mathematical modeling and experiments, we show that metabolic defects caused by clock-environment misalignment result in premature replisome disassembly and replicative abortion in the dark, leaving cells with incomplete chromosomes that persist through the night. Our study thus demonstrates that a major function of this ancient clock in cyanobacteria is to ensure successful completion of genome replication in a cycling environment.

Keywords: DNA replication; cell cycle; circadian clock; cyanobacteria; mathematical modeling.

Fig. 1.

The cyanobacterial circadian clock generates rhythmicity in DNA replication. (A) Schematic diagram showing the entrainment and EdU-labeling procedures. (B) Fluorescence images showing EdU Alexa Fluor 488 foci in wild-type (WT) cells at various $\theta$: subjective dawn ($\theta$ = 0 h), subjective midday ($\theta$ = 6 h), subjective dusk (θ = 12 h), and subjective midnight ($\theta$ = 18 h). Arrows point to EdU foci. (Scale bar = 3 µm.) (C) EdU densities as a function of $\theta$ in WT cells. The values correspond to the number of active replication events per micrometer of cell length during a 10 min pulse-labeling time window centered at each $\theta$. (D) EdU densities as a function of $\theta$ in a clock-deletion strain. (E) Initiation and completion rates inferred using Wiener deconvolution and a 2 h replication time window. All error bars are SEMs from three independent experiments.

Fig. 2.

Circadian clock schedules rhythmic assembly of the replisome. (A) Film strips from time-lapse imaging experiments showing β-EGFP foci in wild-type (WT) background as a function of time in LD cycle condition. (All scale bars = 3 µm.) (B) Average β-EGFP focus density (counts/µm) in WT background in LD as a function of time. Solid lines are seven-point running average, and error bars are SEMs from three independent experiments. (C) Film strips showing β-EGFP and SSB-EGFP foci in WT background as a function of time and $\theta$ in LL condition. (D) Average β-EGFP and SSB-EGFP focus densities as a function of time in LL, under different illumination levels (LOW, MED, and HIGH). ${\tau }_d$ is the doubling time of each condition (mean ± SD). Error bars are SEMs from two to three independent experiments. (E) Average β-EGFP and SSB-EGFP focus densities as a function of $\theta$.

Fig. 3.

The ability to sustain DNA replication in the dark depends on the clock state at the onset of dark. (A) Fluorescence images showing β-EGFP foci at various time points (t) in the dark, for cells subject to a dark pulse either out-of-phase (${\theta }_{\text{dark}}=0$) or in-phase (${\theta }_{\text{dark}}$ = 12) with respect to their entrainments. (Scale bars = 3 µm.) (B) β-EGFP densities as a function of ${\theta }_{\text{dark}}$ during the first 2 h of dark (t = 0, 1, 2 h). (C) The ratio of β-EGFP densities between cells in the subjective nighttime state (12 h ≤ ${\theta }_{\text{dark}}$ < 24 h) and those in the subjective daytime state (0 h ≤ ${\theta }_{\text{dark}}$ < 12 h) when transferred to the dark, plotted as a function of time in the dark. (D) Schematic diagram of the model used to simulate replication events in the dark. (E) Simulated replication events plotted against experimentally collected β-EGFP data in dark. (F) Best-fit initiation decay rate ${\lambda }_{ini}$ and best-fit abortion rate $k_{abort}$ plotted as a function of ${\theta }_{\text{dark}}$. All β-EGFP error bars are SEMs from five independent experiments totaling ∼27,000 cells.

Fig. 4.

Incorrect clock state causes ongoing replication events to abort in the dark. (A, Left) log-survival functions of β-EGFP foci lifetime under various conditions. Solid lines are linear fits through the origin, the negative slopes of which correspond to the apparent dissociation rate constants $\left({\lambda }_{\text{app}\_\text{diss}}\right)$ (Right). Error bars are SEMs bootstrapped from over 2,000 foci trajectories from two to three independent experiments. **P < 0.01 (two-sample t test). (B) SSB-EGFP density time trace in the dark. Error bars are SEMs from two independent experiments. (C) qPCR quantification of ter/oriC ratios in the dark. Error bars are SEMs from four independent experiments. Inset shows ter/oriC ratios averaged throughout the night. ***P < 0.001. (D) Model-predicted ter/oriC ratios assuming 3 to 6 chromosomes per cell (21). (E) dNTP concentrations at subjective dawn and subjective dusk. Error bars are SEMs from four independent experiments. *P < 0.05, **P < 0.01. (F) Fluorescence images showing β-EGFP foci in GalP-expressing cells subject to an out-of-phase dark pulse, without (−) or with (+) glucose supplement. (G) β-EGFP density and apparent dissociation rate constants for GalP-expressing cells. Error bars are SEMs from two (−glucose) or three (+glucose) experiments. (H) Schematic diagram summarizing main findings of this study.