Research ArticleMOLECULAR BIOLOGY

The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks

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Science Advances  08 Apr 2020:
Vol. 6, no. 15, eaaz3327
DOI: 10.1126/sciadv.aaz3327
  • Fig. 1 MGS1 deletion or elimination of Mgs1-ATPase activity reduce the sensitivity of rad5Δ cells to MMS or HU.

    (A) Drop dilution assays. Sensitivity of the strains to chronic treatment with MMS or HU. Serial dilutions (10-fold) of normalized exponentially growing cultures were spotted onto YPD plates containing different amounts of MMS or HU, as indicated, and incubated for 48 hours at 30°C. Strains: Wild type (SY2080), mgs1Δ (YSG15), rad5Δ (SY2214), and mgs1Δrad5Δ (YAJ48) (W303 background). (B) Sensitivity to MMS during S phase. Cells were synchronized in G1 phase with α factor and then released into S phase in medium containing different MMS concentrations. Strains are as in (A). The plots represent the means ± SD from three independent experiments. (C) Drop dilution assays, as in (A). Strains: Wild type (YAJ111), mgs1∆ (YAJ110), mgs1-D31A (YAJ112), mgs1-K183A (YAJ113), rad5∆ (YAJ115), mgs1∆rad5∆ (YAJ114), mgs1-D31A rad5∆ (YAJ116), and mgs1-K183A rad5∆ (YAJ117). (D) Sensitivity to MMS during S phase, as in (B). Strains are as in (C). The plots represent the means ± SD from three independent experiments.

  • Fig. 2 MGS1 deletion in cells lacking Rad5 allows the completion of DNA replication under genotoxic stress conditions.

    (A) PFGE analysis. Cells were synchronized in G1 and released into S phase in medium containing 0.02% MMS and nocodazole for 60 min. The MMS was then removed, and cells were allowed to progress through S phase in medium with nocodazole. Cell cycle progression was monitored by flow cytometry (left top). An ethidium bromide–stained pulse-field gel is shown (right). Chromosomes are labeled with Roman numerals. The quantification of the relative DNA levels of each sample with respect to those in G1 is indicated (left bottom). Strains: Wild type (WT; SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), and mgs1rad5∆ (YAJ48). (B) Suppression of the sensitivity to MMS or HU of rad5∆ cells after Mgs1 elimination is Pol δ–dependent. Drop dilution assays. Serial dilutions (10-fold) of normalized exponentially growing cultures were spotted onto YPD plates containing different amounts of MMS or HU and incubated for 48 hours at 30°C. Strains: Wild type (SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), mgs1rad5∆ (YAJ48), pol3ct (YSG18), pol3ct mgs1∆ (YSG21), pol3ct rad5∆ (YAJ133), and pol3ct mgs1rad5∆ (YAJ135). (C) Reduction of the sensitivity to MMS or HU of rad5∆ cells after Mgs1 elimination requires modification of the K164 residue of PCNA. Drop dilution assays as in (B). Strains: Wild-type MGS1+RAD5+ (SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), mgs1rad5∆ (YAJ48), pol30K164R (TH291), pol30K164R mgs1∆ (YAJ104), pol30K164R rad5∆ (YAJ106), and pol30K164R mgs1rad5∆ (YAJ130).

  • Fig. 3 TLS polymerases have a minor role in the suppression of the sensitivity of rad5∆ cells to MMS or HU when the Mgs1-ATPase activity is eliminated.

    (A) The elimination of the ATPase activity of Mgs1 is compatible with the deletion of RAD5 and the genes encoding TLS polymerases. Examples of tetrad dissection after combining RAD5 deletion in the absence of Mgs1-ATPase activity (mgs1-K183A) with deletions of REV1 (YAJ117 × YAJ96 strains; left), REV3 (YAJ117 × YAJ76; center), or all TLS (REV1, REV3, and RAD30; YAJ183 × YAJ206; right). Spores were grown at 30°C for 48 hours. (B) Drop dilution assays. Serial dilutions (10-fold) of normalized exponentially growing cultures were spotted onto YPD plates containing different amounts of MMS or HU and incubated for 48 hours at 30°C. Strains: Wild-type (YAJ111), mgs1∆ (YAJ110), mgs1-K183A (YAJ113), rad5∆ (YAJ115), mgs1∆rad5∆ (YAJ114), mgs1-K183A rad5∆ (YAJ117), rev1rev3∆rad30∆ (tls∆) (YAJ231), mgs1tls∆ (YAJ233), mgs1-K183A tls∆ (YAJ235), tlsrad5∆ (YAJ237), mgs1-K183A tlsrad5∆ (YAJ230), mgs1-K183A rev3rad5∆ (YAJ165), mgs1-K183A rev1rad5∆ (YAJ196), and mgs1-K183A rad30rad5∆ (YAJ205).

  • Fig. 4 Replication and viability of mgs1rad5∆ cells in the presence of genotoxic stress depend on recombination.

    (A) Mgs1 elimination allows the formation of X structures in rad5∆ cells under DNA-damaging conditions. 2D gel electrophoresis analysis. Samples were taken at the indicated time points after release from G1 arrest in the presence of 0.033% MMS. The genomic DNA was digested with Eco RV–Hind III and analyzed by 2D gel with a probe recognizing the ARS305 early replication origin. Representative autoradiograms of the 2D gels (left) and flow cytometry to monitor cell cycle progression are shown (right). A schematic representation of the main 2D gel signals, the location of the ARS305 probe, and X-molecule quantification are shown. The highest X signal was set as 100%. The red arrows indicate X molecules (recombinants) to compare differences between rad5∆ and mgs1rad5∆ cells. Strains: Wild type (HY1976), mgs1∆ (YAJ319), rad5∆ (YAJ321), and mgs1rad5∆ (YAJ323). (B) Suppression of the sensitivity to MMS or HU of rad5∆ cells by Mgs1 elimination requires Rad52. Drop dilution assays. Serial dilutions (10-fold) of normalized exponentially growing cultures were spotted onto YPD plates containing different amounts of MMS or HU and incubated for 52 hours at 30°C. Strains: Wild type (SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), mgs1rad5∆ (YAJ48), rad52∆ (YAJ11), rad52mgs1∆ (YAJ51), rad52rad5∆ (YAJ138), and rad52mgs1rad5∆ (YAJ140). (C) Suppression of MMS and HU sensitivity of rad5∆ cells when Mgs1 is eliminated requires Rad59. Drop dilution assays as in (B). Strains: Wild type (SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), mgs1rad5∆ (YAJ48), rad59∆ (YCL29), rad59mgs1∆ (YCL30), rad59rad5∆ (YCL31), and rad59mgs1rad5∆ (YCL32).

  • Fig. 5 Esc2 and Elg1 are required for the survival of mgs1rad5∆ cells in the presence of MMS or HU.

    (A) Reduction of the sensitivity to MMS or HU of rad5∆ cells after Mgs1 elimination requires Esc2. Drop dilution assays. Serial dilutions (10-fold) of normalized exponentially growing cultures were spotted onto YPD plates containing different amounts of MMS or HU and incubated for 48 hours at 30°C. Strains: Wild type (SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), mgs1rad5∆ (YAJ48), esc2∆ (YSG436), esc2mgs1∆ (YSG437), esc2rad5∆ (YSG438), and esc2mgs1rad5∆ (YSG442). (B) Suppression of the MMS and HU sensitivity of rad5∆ cells after Mgs1 elimination is Elg1-dependent. Drop dilution assays, as in (A). Strains: Wild type (SY2080), mgs1∆ (YSG15), rad5∆ (SY2214), mgs1rad5∆ (YAJ48), elg1∆ (YAJ71), elg1mgs1∆ (YAJ72), elg1rad5∆ (YAJ73), and elg1mgs1rad5∆ (YAJ74). (C) Genome-wide binding pattern of Elg1 by ChIP-on-chip after release of cells from G1 block in medium containing 0.02% MMS for 30 min. The histogram peaks on the y axis depict the genome browser view of Elg1-Flag binding represented as the average signal ratio in log2 scale of loci enriched in the immunoprecipitated fraction along the indicated regions. The x axis shows chromosomal coordinates. The P values relate to the genome-wide overlap among Elg1 clusters in the different strains. Chromosome VI is shown as an example. The location of some replication origins is indicated. Strains: ELG1-10FLAG (wild type, HY1976), ELG1-10FLAG mgs1∆ (YAJ319), ELG1-10FLAG rad5∆ (YAJ321), and ELG1-10FLAG mgs1rad5∆ (YAJ323). (D) Genome-wide binding pattern of Pol3 by ChIP-on-chip. Experimental conditions and picture details are as in (C). Strains: POL3-3FLAG (wild type, YAJ347), POL3-3FLAG mgs1∆ (YAJ348), POL3-3FLAG rad5∆ (YAJ349), and POL3-3FLAG mgs1rad5∆ (YAJ350).

  • Fig. 6 Model for DDT during chromosomal replication, in the presence or absence of Rad5 or Mgs1.

    (A) MGS1+RAD5+ cells. DNA damage is tolerated predominantly by error-free Rad5-dependent template switching during chromosome replication (27). Recombination is globally inhibited during replication, at ongoing forks, by recruitment of the Srs2 antirecombinase helicase to SUMO-PCNA (10, 11), but recombination-mediated damage bypass by template switching is locally allowed, at damaged stalled forks, by the SUMO-like domain protein Esc2, which counteracts Srs2 (18). Esc2 binds at sites of stalled replication and promotes Elg1 binding at forks, which, in turn, induces regulated unloading of Srs2 bound to SUMO-PCNA by Elg1 and degradation of the helicase by Slx5-Slx8–mediated proteasome-dependent degradation, thus reducing Srs2 levels locally (18). RPA, replication protein A. (B) mgs1rad5∆ cells. In the absence of Rad5, Mgs1 elimination allows DNA damage bypass at sites of perturbed replication. The absence of Mgs1 in cells lacking Rad5 facilitates a recombination-driven replication mechanism that is Rad52, Rad59, and Pol δ dependent. As in the template switching pathway used by MGS1+RAD5+ cells (A), this salvage pathway of recombination requires Esc2 and Elg1 and, therefore, is very likely conducted in the same manner. HR, homologous recombination. (C) MGS1+rad5∆ cells. The absence of Rad5 impedes DNA damage bypass by template switching, which causes forks block, and cells die because replication cannot be completed (2729) as an alternative mechanism to overcome replication perturbations is prevented. This correlates with the presence of Mgs1 and the accumulation of Elg1 at stalled forks, which might reflect SUMO-PCNA stabilization. See details in the main text.

Supplementary Materials

  • Supplementary Materials

    The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks

    Alberto Jiménez-Martín, Irene Saugar, Chinnu Rose Joseph, Alexandra Mayer, Carl P. Lehmann, Barnabas Szakal, Dana Branzei, José Antonio Tercero

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