doi: 10.1371/journal.pgen.1011917.
eCollection 2025 Oct.
Stn1 supports Mec1 function in protecting stalled replication forks from degradation
Affiliations
- PMID: 41091836
- PMCID: PMC12548912
- DOI: 10.1371/journal.pgen.1011917
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Stn1 supports Mec1 function in protecting stalled replication forks from degradation
PLoS Genet.
.
Abstract
Replication stress threatens genome integrity by exposing replication forks to nucleolytic degradation. In both yeast and humans, the checkpoint kinases Mec1 and Rad53 limit deleterious single-stranded DNA (ssDNA), yet the protective mechanisms remain incompletely defined. Here, we identify a role for the CST subunit Stn1 in cooperating with Mec1 to restrain ssDNA formation under nucleotide depletion. A gain-of-function allele (stn1-L60F) suppresses the sensitivity to replication stress of Mec1-deficient cells and reduces ssDNA at stalled replication forks, whereas a loss-of-function truncation (stn1-ΔC) exacerbates both phenotypes. Mechanistically, Stn1 opposes the resection activities of Mre11, Exo1, and Sgs1 by promoting Polα-primase-dependent fill-in and by limiting their association with stalled replication forks, with the latter mechanism predominating in the suppression exerted by Stn1L60F. Thus, Stn1 works with the checkpoint to curb nuclease activity at sites of replication stress.
Copyright: © 2025 Casari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
(A) Exponentially growing cell cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without HU. (B) Cells were arrested in G1 with α-factor (time zero) and then released into YEPD containing 0.2M HU. Aliquots were removed from the HU-treated cultures at timed intervals to score for colony‐forming units on YEPD plates at 25°C. Plotted values are the mean values ± s.d. from three independent experiments. (C) Exponentially growing cell cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without HU. (D) Western blot analysis with an anti-HA antibody of protein extracts prepared from exponentially growing cells. The same amount of extracts was stained with Coomassie Blue as loading control. (E) Cells were arrested in G1 with α-factor (time zero) and then released into YEPD containing 0.2M HU. Protein extracts prepared at different time points after α-factor release were analyzed by western blot using an anti-Rad53 antibody. (F) Quantitative analysis of Rad53 phosphorylation shown in panel (E) was performed by calculating the ratio of band intensities for slowly-migrating bands to the total amount of protein. Plotted values are the mean values ± s.d. from three independent experiments. ***p < 0.005, **p < 0.01, *p < 0.05 (Student’s t-test). (G) Phleomycin (10 μg/mL) was added to exponentially growing cells, and protein extracts were analyzed by western blot using an anti-Rad53 antibody. (H) Quantitative analysis of Rad53 phosphorylation shown in panel (G) was performed as in panel (F).
Exponentially growing YEPD cell cultures were arrested in G1 with α-factor (time zero) and then released into YEPD containing 0.2M HU. Genomic DNA prepared at different time points after α-factor release was either digested or mock-digested with SspI and used as a template in qPCR. The value of SspI-digested over non-digested DNAs was determined for each time point after normalization to an amplicon on chromosome XI that does not contain SspI sites. The data shown are expressed as fold-enrichments in ssDNA at different time points after α-factor release in HU relative to the α-factor (time zero) (set to 1.0). A locus containing SspI sites on chromosome XI is used as a control (control locus). Plotted values are the mean values ± s.d. from three independent experiments. ***p<0.005, **p<0.01, *p<0.05 (Student’s t-test).
Exponentially growing cell cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without HU.
Exponentially growing cell cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without HU.
Exponentially growing YEPD cell cultures were arrested in G1 with α-factor (time zero) and then released into YEPD containing 0.2M HU. ssDNA at different distances from ARS607 was assessed as described in Fig 2. ***p < 0.005, **p < 0.01, *p < 0.05 (Student’s t-test).
(A) ChIP analysis of Stn1-HA and Stn1-L60F-HA. Cells were arrested in G1 with α-factor and then released into YEPD containing 0.2M HU at time zero. Relative fold-enrichment of HA-tagged Stn1 and Stn1-L60F at ARS305 and ARS607 replication origins was determined after ChIP with an anti-HA antibody and qPCR analysis. Plotted values are the mean values ± s.d. from three independent experiments. ***p < 0.005, **p < 0.01, *p < 0.05 (Student’s t-test). (B) Domain architecture of CST components. Cdc13 contains four OB-fold domains: Cdc13-terminal (Cdc13 N), which binds Pol1, OB2, the DNA-binding domain or OB3 (DNAB), and OB4, which binds Stn1. Stn1 comprises an N-terminal OB-fold domain (DNAB), which binds DNA, and two C-terminal winged helix-turn-helix motifs (WH1 and WH2). The C-terminal region of Stn1 interacts with Cdc13 (via WH2) and with Pol12 (via WH1). Ten1 consists of a single OB-fold domain that binds the N-terminal OB-fold/DNA-binding domain of Stn1. Red arrows indicate protein-protein interactions. The domains are in orange except for the DNA-binding domains, in blue. (C) AlphaFold 3-predicted model of the yeast CST complex bound to a 20-nt ssDNA (black ribbon). The Cdc13 N domain is not depicted because its position is variable with respect to the rest of the complex (S3 Fig). (D) Detail of the ssDNA-binding interface in the HADDOCK 2.4 water refinement top-scoring model for wild-type CST complex. (E) Detail of the ssDNA-binding interface in the HADDOCK 2 water-refined top-scoring model for the mutant CSL60FT complex.
(A) Exponentially growing YEPD cell cultures were arrested in G1 with α-factor (time zero) and then released into YEPD containing 0.2M HU. ssDNA at different distances from ARS607 was assessed as described in Fig 2. ***p < 0.005, **p < 0.01, *p < 0.05 (Student’s t-test). (B) Exponentially growing cell cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without HU.
Cells were arrested in G1 with α-factor and then released into YEPD containing 0.2M HU at time zero. Relative fold-enrichment of Myc-tagged Mre11, Myc-tagged Exo1 and HA-tagged Sgs1 at ARS305 and ARS607 replication origins was determined after ChIP with an anti-Myc or an anti-HA antibody and qPCR analysis. Plotted values are the mean values ± s.d. from three independent experiments. ***p < 0.005, **p < 0.01, *p < 0.05 (Student’s t-test).
(A) Exponentially growing YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG, followed by Stn1-HA and Stn1-L60F-HA ChIP at the indicated distances from the HO-cut site compared to untagged Stn1 (no tag). Data are expressed as fold-enrichment at the HO-cut site over that at a non-cleavable locus (ARO1), after normalization to the corresponding input for each time point. Fold-enrichment was then normalized to cut efficiency. Plotted values are the mean values ± s.d. from three independent experiments. *p < 0.05 (Student’s t-test). (B) YEPR exponentially growing cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero. Western blot analysis with an anti-Rad53 antibody of protein extracts from samples taken at the indicated times after HO induction. (C) Quantification of ssDNA by qPCR at the indicated distances from the HO cut site. Plotted values are the mean values ± s.d. from three independent experiments. **p < 0.01, *p < 0.05 (Student’s t-test). (D) Exponentially growing YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG to induce HO expression. Relative fold-enrichment of Mre11-Myc, Exo1-Myc and Sgs1-HA at the HO-induced DSB was evaluated after ChIP with anti-Myc or anti-HA antibody and qPCR. Plotted values are the mean values ± s.d. from three independent experiments. **p < 0.01, *p < 0.05 (Student’s t-test).
dNTP depletion stalls replication forks. When Mec1 is compromised (mec1-100), the CMG helicase keeps unwinding, leading to excessive accumulation of incomplete Okazaki fragments, depletion of replication factors, aberrant fork restart, and exposure of DNA to nuclease attack. When Stn1 is present, Polα-primase-mediated fill-in synthesis is promoted and the fork-proximal recruitment/retention of Mre11, Exo1, and Dna2-Sgs1 on DNA is restrained, enabling restart. The gain-of-function stn1-L60F mutation increases Stn1 association with stalled replication fork, thereby limiting the association of Mre11, Exo1, and Dna2-Sgs1 and the resulting ssDNA generation more effectively than wild-type Stn1. In contrast, the loss-of-function stn1-ΔC mutation fails to restrain the resection activity of Mre11, Exo1, and Dna2, leading to extensive DNA degradation and replication failure. A limited nuclease activity remains beneficial for fork remodeling in mec1-100. In wild-type cells, checkpoint signaling restrains CMG-driven unwinding, prevents excessive Okazaki-fragment formation, preserves replication factors, and permits proper DNA replication restart.
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