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, 19 (24), 3055-69

Replisome Instability, Fork Collapse, and Gross Chromosomal Rearrangements Arise Synergistically From Mec1 Kinase and RecQ Helicase Mutations

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Replisome Instability, Fork Collapse, and Gross Chromosomal Rearrangements Arise Synergistically From Mec1 Kinase and RecQ Helicase Mutations

Jennifer A Cobb et al. Genes Dev.

Abstract

The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1Delta with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1Delta mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1Delta mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1Delta cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.

Figures

Figure 1.
Figure 1.
Highly synergistic effects of mec1-100 and sgs1Δ mutations on chromosome stability and recovery from HU. (A) Drop assays on YPD ± 10 mM HU were performed with exponentially growing cultures of the indicated W303-1a (GA-180) derivates, using a 1:5 serial dilution series: sgs1Δ (GA-1761), mec1-100 (GA-2474), mec1-100 sml1Δ (GA-2478), mec1-100 sgslΔ (GA-2514), or mec1Δ sml1Δ (GA-2895). (B) Cell viability was monitored as colony outgrowth from cultures synchronized by α-factor and held in YPD + 0.2 M HU for indicated times.
Figure 2.
Figure 2.
Replication fork collapse in mec1-100 sgs1Δ mutants. For all panels, cells were grown in minimal medium with heavy (H) isotopes and blocked in G1 with α-factor. The cultures were held in α-factor for an additional 30 min in light (L) isotope before dividing and releasing into fresh medium ± 0.2 M HU. Samples were taken at 40, 80, and 120 min, when cells were released from HU and their recovery was monitored. DNA content as determined by flow cytometry and cell viability for all strains was scored at the indicated time points. A time course of DNA replication at ARS607 was analyzed by density transfer after release from α-factor arrest into medium with 0.2 M HU, using specific probes recognizing the ClaI/SalI fragments 1 and 2. The relative amounts of radioactivity in the hybridized DNA are plotted against the gradient fraction number. The positions of unreplicated (heavy-heavy, HH) and fully replicated heavy-light (HL) are indicated. At later time points, the position of the initial HH peak is shown for comparison (gray area). Corresponding FACS analysis and survival assays are shown for each isogenic strain bearing the following mutations: wild-type (YJT110) (A); sgs1Δ (YVR1) (B); mec1-100 (GA-2931) (C); and mec1-100 sgs1Δ (GA-2930) (D).
Figure 3.
Figure 3.
Loss of DNA polymerases at stalled replication forks in mec1-100 sgs1Δ cells. (A) Neutral/neutral 2D gel analysis was performed at ARS607 in wild-type (GA-1020) and mec1-100 sgs1Δ (GA-2514) cells released from α-factor arrest into YPD + 0.2 M HU. Genomic DNA was prepared from cells collected at 0 (G1) or 20 min after release, and the Southern transfer was probed with a 2.6-kb fragment spanning ARS607. (B) Primers were designed to amplify genomic regions on Chr 6 corresponding to early-firing origin ARS607 (filled symbols) and a nonorigin site, +14 kb (open symbols). ChIP was performed on cultures synchronized in G1 by α-factor arrest, and released into pre-warmed YPD + 0.2 M HU, prior to fixation with 1% formaldehyde at the indicated time points. (C) ChIP with anti-Myc (9E10) is used to quantify Myc-Orc2 presence at ARS607 in isogenic wild-type (GA-2897, diamonds) and mec1-100 sgs1Δ (GA-2896, ovals) cells. (D-G) ChIP with anti-Myc (9E10) or anti-HA (12CA5) precipitated HA-tagged DNA pol α (squares) or Myc-tagged DNA pol ε (diamonds). The strains used were wild-type strains GA-2238 and GA-2448 in D; sgs1Δ strains GA-2256 and GA-2450 in E; mec1-100 strains GA-2567 and GA-2515 in F; and mec1-100 sgs1Δ strains GA-2578 and GA-2516 in G. In E-G, wild-type signals are shown as light-gray dashed lines for comparison. Controls and quantitation are described in Materials and Methods. Standard deviation is calculated from duplicate runs and multiple independent experiments.
Figure 4.
Figure 4.
Rad53 is needed to stabilize MCM proteins but not DNA polymerases at stalled forks. (A) ISA analysis of Rad53 autophosphorylation was performed on wild-type (GA-1020), rad53-11 (GA-2240), mec1-100 (GA-2474), and mec1-100 sgs1Δ (GA-2514) cells. For each strain, the upper box shows the incorporation of γ32-ATP into Rad53, and the bottom panel shows the same blot probed with anti-RNaseH42 to normalize loading (*). Time (in minutes) after α-factor release is indicated above each panel, and “std” is 5 μL of a standard containing a known amount of a HU-activated Rad53. For every sample, protein concentration was determined by Coomassie blue staining prior to equally loading gels. Dried filters were exposed for equal times on a Bio-Rad PhosphorImager, before reprobing for RNaseH42 to normalize signals. (B) Quantification of Rad53 autophosphorylation displayed as a normalized percentage of std. Shown is an average of two experiments with standard deviations between 5% and 15%. (C,D) ChIP was performed as described in Figure 3 for HA-tagged DNA pol α (squares) and Myc-tagged DNA pol ε (diamonds) in rad53-11 strain GA-2574 and rad53-11 sgs1Δ strain GA-2576. (E,F) ChIP was performed for Myc-tagged Mcm7 (diamonds) in cultures released from α-factor into 0.2 M HU as in Figure 3 using wild type (GA-1003) and rad53-11 (GA-3054). rtPCR was performed as described for ARS607 (filled symbols) and +14 kb (open symbols). Wild-type and sgs1Δ signals are shown in light-gray dashed lines for comparison.
Figure 5.
Figure 5.
Rpa1 is displaced from stalled replication forks in mec1-100 sgs1Δ cells. ChIP was performed on Myc-tagged Rpa1 (squares) in cultures release from α-factor into 0.2 M HU as described in Figure 3 using the following strains: wild-type (GA-1113) (A), sgs1Δ (GA-2439) (B), mec1-100 (GA-2571) (C), mec1-100 sgs1Δ (GA-2581) (D), and mec1Δ sml1Δ (GA-2582) (E). rtPCR-amplified regions correspond to ARS607 (filled symbols) and +14 kb (open symbols), with the wild-type signal for Rpa1 shown as a dashed line. From the same experiment, the level of Myc-Rpa1 at the late origin ARS501 is shown for indicated wild-type and mutant strains.
Figure 6.
Figure 6.
Ddc2 recruitment drops in sgs1Δ but not mec1-100 strains. ChIP was performed on HA-tagged Ddc2 (diamonds) in cultures released from α-factor into 0.2 M HU exactly as described in Figure 3 using the following strains: wild-type (GA-2462) (A), mec1Δ (GA-2463) (B), mec1-100 (GA-2475) (C), and sgs1Δ (GA-2519) (D). rtPCR-amplified regions correspond to ARS607 (filled symbols) and +14 kb (open symbols), with the wild-type signal for Ddc2 shown as a dashed line. From the same experiment, the level of Ddc2 at the late origin ARS501 is shown for indicated wild-type and mutant strains (E).
Figure 7.
Figure 7.
Modification of H2A at replication forks is Mec1-specific. (A) Primers as previously described in Figure 3 were used to amplify the early-firing origin ARS607 (stippled) and an origin-proximal site (+4 kb; white), or late-firing origin ARS501 (black). (B) ChIP was performed on a wild-type (GA-2448) culture as described in Figure 3, released from α-factor into 0.2 M HU, using a phospho-specific rabbit polyclonal antibody recognizing the Ser 129-P H2A epitope (a gift from W. Bonner). Myc-tagged DNA pol ε was precipitated in parallel (data not shown). (C) ChIP for H2A-P as in B was performed on a wild-type culture following synchronous release from pheromone arrest into YPD at 16°C, in the absence of HU. (D) ChIP as described in B except that the strains used were mec1Δ (GA-2588) and tel1Δ (GA-2002). Here the ratio of absolute fold enrichments is reported after the rtPCR signals are normalized to a wild-type control in duplicate independent experiments: The scaling factor is 1.00 for mec1Δ and 0.268 for tel1Δ. Error bars were similar for both wild-type and mutant strains.
Figure 8.
Figure 8.
Mec1/Ddc2 and Sgs1 stabilize RPA and DNA polymerases at stalled forks. This model summarizes the pathways that stabilize the replisome in cells exposed to HU. Sgs1, like large T antigen, is proposed to provoke a conformational change in RPA that promotes stable binding of DNA pol α as a primosome. Mec1/Ddc2 kinase also acts on Mrc1 to stabilize polymerases, while Rad53 either uncouples or displaces the MCM complex.

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