Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase
- Wellcome Trust/Cancer Research United Kingdom Gurdon Institute and Department of Biochemistry, University of Cambridge, United Kingdom
Figures
Figure 1 with 1 supplement
Dbf4 and Sld3 are phosphorylated by Rad53 after DNA damage in G1 and G2 phase.
(A) Flow cytometry of strains arrested in G1 phase with the mating pheromone alpha factor. Strains were held in G1 phase, with or without the addition of 10 μg/ml 4-NQO for the indicated times. All …
Figure 1—figure supplement 1
Sld3 phosphorylation in G1 phase after DNA damage is Rad53 dependent.
(A) Flow cytometry of strains arrested in G1 phase with the mating pheromone alpha factor. Strains were held in G1 phase, with the addition of 10 μg/ml 4-NQO for the indicated times. All strains are …
Figure 2 with 1 supplement
Rad53 phosphorylates Sld3 and Dbf4 in G1 and G2 phases at the same residues as in S-phase (A and B) as Figure 1B,C.
sld3-A and dbf4-19A refer to mutant alleles with Rad53 phosphorylation sites mutated to alanine (38 sites for Sld3 and 19 sites for Dbf4). (C) As Figure 1E, except this is western blot from an …
Figure 2—figure supplement 1
Dbf4-4A is expressed at similar levels to wild type and is also refractory to Rad53 phosphorylation in G1 phase.
Western blot of strains arrested in G1 phase with the mating pheromone alpha factor. Strains were held in G1 phase, with the addition of 10 μg/ml 4-NQO for the indicated times. All strains are bar1∆ …
Figure 3 with 1 supplement
Checkpoint-dependent inhibition of origin firing prevents re-replication in G2 phase.
(A) Flow cytometry of the indicated strains grown overnight in YPraffinose, then arrested in G2/M with nocodazole. After addition of fresh nocodazole, 2% galactose was added for the indicated times …
Figure 3—figure supplement 1
Checkpoint-dependent inhibition of origin firing prevents re-replication in G2 phase.
(A) Growth assay of the indicated strains. (B) Analysis of mitochondrial DNA content by sequencing. (C) Comparison of DNA copy number at all origins between the strain over-expressing the …
Figure 4 with 3 supplements
Checkpoint-dependent inhibition of origin firing prevents premature replication initiation at all origins at the G1–S transition.
(A) Activation of Rad53 in G1 phase can delay genome duplication by at least two mechanisms; by inhibition of the transcriptional activator Swi6, which is required for G1- and subsequently S-phase …
Figure 4—figure supplement 1
4-NQO addition in G1 phase delays the G1/S transition.
(A) Flow cytometry of the indicated strains, which were grown overnight in YPraffinose and arrested in G1 phase with alpha factor (G1 time point). Strains were then held in G1 phase for an extra 30 …
Figure 4—figure supplement 2
Rad53 activation in G1 phase inhibits all origin firing through phosphorylation of Sld3 and Dbf4.
Replication profile of chromosome VII as an example at 20, 30, and 40 min from the experiment in Figure 4B. Green * are examples of very early firing origins, which initiate at the 20 min time point …
Figure 4—figure supplement 3
Genetic interactions of the sld3-A dbf4-A alleles with mutants that either accelerate or delay the G1/S transition.
Scatter plot of the fitness of the yeast genome knock out collection grown in 0.5 μg/ml phleomycin with (y-axis) or without (x-axis) the sld3-A dbf4-A alleles. Each dot corresponds to a different …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Rad53, rabbit polyclonal | Abcam | (ab104232) | Antibody (1:5000) |
| Antibody | Anti-c-myc, mouse monoclonal | Merck Life Science | (11667149001) | Antibody (1:1000) |
| Peptide, recombinant protein | Alpha-factor | GenScript | (RP01002) | Peptide |
| Commercial assay or kit | TruSeq Nano DNA Low Throughput Library Prep Kit | Illumina | (20015964) | Commercial assay or kit |
| Chemical compound, drug | Phos-tag Acrylamide AAL-107 | Alpha Laboratories | (304–93521) | Chemical |
| Chemical compound, drug | Nocodazole | Merck Life Science | (M1404) | Chemical |
| Chemical compound, drug | 4-Nitroquinoline N-oxide | Merck Life Science | (N8141) | Chemical |
| Software, algorithm | LocalMapper software | Batrakou et al., 2020 | ||
| Software, algorithm | Repliscope software | Batrakou et al., 2020 |
