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, 8 (11), e81015
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A Rad53 Independent Function of Rad9 Becomes Crucial for Genome Maintenance in the Absence of the Recq Helicase Sgs1

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A Rad53 Independent Function of Rad9 Becomes Crucial for Genome Maintenance in the Absence of the Recq Helicase Sgs1

Ida Nielsen et al. PLoS One.

Abstract

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Efficient recovery from MMS reveals a strong synergistic functionality between Sgs1/Top3 and Rad9.
(A) Survival was monitored as described in Materials and Methods after 70 min exposure to different concentrations of MMS for the indicated strains: wild type (LBy-1), sgs1Δ (LBy-129), top3Δ (LBy-7), rad9Δ (LBy-316), sgs1Δ rad9Δ (LBy-44) and top3Δ rad9Δ (LBy-27). (B) Survival as in A for isogenic strains rad24Δ (LBy-391), sgs1Δ rad24Δ (LBy-36) and top3Δ rad24Δ (LBy-28). The wild type, sgs1Δ and top3Δ survival curves are added in for comparison from A.
Figure 2
Figure 2. sgs1Δ induced genomic instability increases dramatically in rad9Δ cells as measured by Gross Chromosomal Rearrangements.
(A) GCR were measured after exposure to 0.02% MMS, which results in 10% survival rate for the sgs1Δrad9Δ strain. GCR is shown as fold increase over wild type for the following strains: sgs1Δ (LBy-388), rad24Δ (LBy-406), rad9Δ (LBy-389), sgs1Δrad9Δ (LBy-400), sgs1Δrad24Δ (LBy-407). (B) Spontaneous GCR was measured for wild type (LBy-383), sgs1Δ (LBy-388), rad9Δ (LBy-389) and sgs1Δrad9Δ (LBy-400). GCR is shown as fold increase over wild type.
Figure 3
Figure 3. Rad9 works parallel to Sgs1/Top3 in the intra-S phase checkpoint response induced by MMS.
(A) Experimental outline. (B) ISA analysis of Rad53 auto-phosphorylation measured for wild type (LBy-1), sgs1Δ (LBy-129), top3Δ (LBy-7) and sgs1Δtop3Δ (LBy-8). Cells were synchronised in G1, released into 0.02% MMS, and analysed at indicated times by ISA. For each strain the upper box shows the incorporation of γ32-ATP into Rad53, and the bottom panel a Western for RnaseH42 on the same blot (*). Time (min) after alpha-factor release is indicated above each panel. Std is 5 µl of a sample containing a fixed amount of activated Rad53 standard, which is used to normalise all gels after identical exposure times (see Materials and Methods). FACS samples were taken at 15 min intervals at the beginning of the experiment and 30 min intervals at the end of the experiment and shown on the right. (C) As in B but with the following strains: rad9Δ (LBy-316), sgs1Δrad9Δ (LBy-44) and top3Δrad9Δ (LBy-27). (D) As in B but with the following strains: rad24Δ (LBy-391), sgs1Δrad24Δ (LBy-36), top3Δrad24Δ (LBy-28) and top3Δsgs1Δrad24Δ (LBy-40).
Figure 4
Figure 4. Chk1 activation is equally compromised in rad24Δ and rad9Δ cells.
Chk1 upshift assay were performed to investigate checkpoint activation for the following strains: wild type (LBy-366), sgs1Δ (LBy-372), rad9Δ (LBy-374) and rad24Δ (LBy-390). Synchronized cultures of cells were released into S phase in the presence of 0.1% of MMS and aliquots were taken at the indicated times for analysis.
Figure 5
Figure 5. The Rad53 checkpoint function of Rad9 is not required for GCR suppression and growth in the presence of MMS in cells lacking Sgs1.
(A) Illustration of the domain structure of Rad9. (B) Immunoprecipitations were conducted with extracts from the contructed rad9 7xA-HA strain (LBy-471) and the sgs1Δrad9 7xA-HA strain (LBy-472) to verify the 7xA mutations. Immunoprecipitations were performed with anti-HA antibody in the presence and absence of MMS. (C) Survival was monitored as described in Materials and Methods after 70 min exposure to different concentrations of MMS for the indicated strains: wild type (LBy-1), sgs1Δ (LBy-129), rad9Δ (LBy-316), sgs1Δrad9Δ (LBy-44), rad9 7xA (LBy-471), sgs1Δ rad9 7xA (LBy-472). (D) GCR were measured after exposure to 0.02% MMS and is shown as fold increase over wild type for the isogenic strains: sgs1Δ (LBy-388), rad24Δ (LBy-406), rad9Δ (LBy-389), rad9 7xA (LBy-473), sgs1Δ rad9 7xA (LBy-474).

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References

    1. Khakhar RR, Cobb JA, Bjergbaek L, Hickson ID, Gasser SM (2003) RecQ helicases: multiple roles in genome maintenance. Trends Cell Biol 13: 493–501. - PubMed
    1. Mohaghegh P, Hickson ID (2002) Premature aging in RecQ helicase-deficient human syndromes. Int J Biochem Cell Biol 34: 1496–1501. - PubMed
    1. Gangloff S, McDonald JP, Bendixen C, Arthur L, Rothstein R (1994) The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol Cell Biol 14: 8391–8398. - PMC - PubMed
    1. Watt PM, Hickson ID, Borts RH, Louis EJ (1996) SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae. Genetics 144: 935–945. - PMC - PubMed
    1. Frei C, Gasser SM (2000) The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci. Genes Dev 14: 81–96. - PMC - PubMed

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Grant support

This work was supported by grants from the Danish Research Council (FNU 272-07-0366), and Aase og Einar Danielsens Foundation to L.B., and the Danish Cancer Society to A.H.A. and L.B. I.B.B. is supported by the Lundbeckfoundation (53/06). S.M.G. was supported by a Swiss Cancer League grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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