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

<div><p>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 <i>SGS1</i> and <i>RAD9</i> in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between <i>SGS1</i> and <i>RAD24</i>. 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 <i>SGS1</i> and <i>RAD9</i> for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for <i>SGS1</i> and <i>RAD24</i>. 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.</p></div

Rad9 works parallel to Sgs1/Top3 in the intra-S phase checkpoint response induced by MMS.

<p>(<b>A</b>) Experimental outline. (<b>B</b>) ISA analysis of Rad53 auto-phosphorylation measured for wild type (LBy-1), <i>sgs1Δ</i> (LBy-129), <i>top3Δ</i> (LBy-7) and <i>sgs1Δtop3Δ</i> (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081015#s2" target="_blank">Materials and Methods</a>). 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. (<b>C</b>) As in B but with the following strains: <i>rad9Δ</i> (LBy-316), <i>sgs1Δrad9Δ</i> (LBy-44) and <i>top3Δrad9Δ</i> (LBy-27). (<b>D</b>) As in B but with the following strains: <i>rad24Δ</i> (LBy-391), <i>sgs1Δrad24Δ</i> (LBy-36), <i>top3Δrad24Δ</i> (LBy-28) and <i>top3Δsgs1Δrad24Δ</i> (LBy-40).</p

<i>Saccharomyces cerevisiae</i> strains used in this study.

<p><i>Saccharomyces cerevisiae</i> strains used in this study.</p

Chk1 activation is equally compromised in <i>rad24Δ</i> and <i>rad9Δ</i> cells.

<p>Chk1 upshift assay were performed to investigate checkpoint activation for the following strains: wild type (LBy-366), <i>sgs1Δ</i> (LBy-372), <i>rad9Δ</i> (LBy-374) and <i>rad24Δ</i> (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.</p

The Rad53 checkpoint function of Rad9 is not required for GCR suppression and growth in the presence of MMS in cells lacking Sgs1.

<p>(<b>A</b>) Illustration of the domain structure of Rad9. (<b>B</b>) Immunoprecipitations were conducted with extracts from the contructed <i>rad9 ^7xA-</i>HA strain (LBy-471) and the <i>sgs1Δrad9 ^7xA</i>-HA strain (LBy-472) to verify the 7xA mutations. Immunoprecipitations were performed with anti-HA antibody in the presence and absence of MMS. (<b>C</b>) Survival was monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081015#s2" target="_blank">Materials and Methods</a> after 70 min exposure to different concentrations of MMS for the indicated strains: wild type (LBy-1), <i>sgs1Δ</i> (LBy-129), <i>rad9Δ</i> (LBy-316), <i>sgs1Δrad9Δ</i> (LBy-44), <i>rad9 ^7xA</i> (LBy-471), <i>sgs1Δ rad9 ^7xA</i> (LBy-472). (<b>D</b>) GCR were measured after exposure to 0.02% MMS and is shown as fold increase over wild type for the isogenic strains: <i>sgs1Δ</i> (LBy-388), <i>rad24Δ</i> (LBy-406), <i>rad9Δ</i> (LBy-389), <i>rad9 ^7xA</i> (LBy-473), <i>sgs1Δ rad9 ^7xA</i> (LBy-474).</p

<i>sgs1Δ</i> induced genomic instability increases dramatically in <i>rad9Δ</i> cells as measured by Gross Chromosomal Rearrangements.

<p>(<b>A</b>) GCR were measured after exposure to 0.02% MMS, which results in 10% survival rate for the <i>sgs1Δrad9Δ</i> strain. GCR is shown as fold increase over wild type for the following strains: <i>sgs1Δ</i> (LBy-388), <i>rad24Δ</i> (LBy-406), <i>rad9Δ</i> (LBy-389), <i>sgs1Δrad9Δ</i> (LBy-400), <i>sgs1Δrad24Δ</i> (LBy-407). (<b>B</b>) Spontaneous GCR was measured for wild type (LBy-383), <i>sgs1Δ</i> (LBy-388), <i>rad9Δ</i> (LBy-389) and <i>sgs1Δrad9Δ</i> (LBy-400). GCR is shown as fold increase over wild type.</p

Efficient recovery from MMS reveals a strong synergistic functionality between Sgs1/Top3 and Rad9.

<p>(<b>A</b>) Survival was monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081015#s2" target="_blank">Materials and Methods</a> after 70 min exposure to different concentrations of MMS for the indicated strains: wild type (LBy-1), <i>sgs1Δ</i> (LBy-129), <i>top3Δ</i> (LBy-7), <i>rad9Δ</i> (LBy-316), <i>sgs1Δ rad9Δ</i> (LBy-44) and <i>top3Δ rad9Δ</i> (LBy-27). (<b>B</b>) Survival as in A for isogenic strains <i>rad24Δ</i> (LBy-391), <i>sgs1Δ rad24Δ</i> (LBy-36) and <i>top3Δ rad24Δ</i> (LBy-28). The wild type, <i>sgs1Δ</i> and <i>top3Δ</i> survival curves are added in for comparison from A.</p

Imprecise end joining increased in cells with Xrs2 FHA domain mutations in a Tel1-dependent manner.

<p>A. System used to examine imprecise end joining. Two HO-cutting sites were introduced in opposite orientations within the <i>MAT</i> locus on chromosome III, that result in two kinds of DSB ends: non-complementary single-stranded over hangs (Ura⁻) and complementary overhangs (Ura⁺), which are repaired by imprecise end joining and precise end joining, respectively. B. Xrs2 protein domains for wild-type and mutant <i>Saccharomyces cerevisiae</i> (sc) proteins. Mre11 BD, Mre11-binding domain; Tel1 BD, Tel1-binding domain. C. Frequencies of Ura⁻ and Ura⁺ cells indicate imprecise end joining and precise end joining, respectively, in wild-type (SLY19), <i>xrs2-314M</i> (DIY002), <i>xrs2-SH</i> (DIY016) and <i>xrs2</i>Δ (DIY007) strains. D. End-joining frequencies in wild-type, <i>tel1</i>Δ (DIY129), <i>tel1</i>Δ <i>xrs2-314M</i> (DIY131) and <i>tel1</i>Δ <i>xrs2-SH</i> (DIY134) strains assessed as in C. E. End-joining frequencies in wild-type, <i>sae2</i>Δ (DIY059), <i>sae2</i>Δ <i>xrs2-314M</i> (DIY062) and <i>sae2</i>Δ <i>xrs2-SH</i> (DIY065) strains assessed as in C. F. End-joining frequencies in wild-type (SLY19), <i>yku70</i>Δ (DIY033), <i>yku70</i>Δ <i>xrs2-314M</i> (DIY051) and <i>yku70</i>Δ <i>xrs2-SH</i> (DIY048) strains assessed as in C. G. End-joining frequencies in wild-type, <i>xrs2-SH</i> (DIY134), <i>lif1-SST</i> (DIY027), <i>lif1-T113A</i> (DIY072), <i>lif1-SST xrs2-SH</i> (MSY5655), <i>lif1-T113A xrs2-SH</i> (MSY5652) and <i>lif1</i>Δ (KMY691) strains assessed as in C. The wild-type data in C, D and E are from a single experiment. Data are presented as the mean ± SD from three independent experiments. Significance was calculated with a Student’s <i>t</i>-test: **<i>p</i><0.001; *<i>p</i><0.05; n.s., not significant.</p

Recruitment of DDR proteins to non-complementary DSB ends.

<p>A. Construction of DSB sites at the MAT locus on chromosome III of strain SLY19 and position of the primer pair for qPCR for ChIP analysis and QAOS assay. B. The association of Xrs2 protein with DSBs in SLY19 (–Tag) or in its derivatives with <i>XRS2-13Myc</i> (wild type, DIY109; <i>xrs2-314M</i>, DIY116; <i>xrs2-SH</i>, DIY106). Error bars show the SD from three independent qPCR trials. C. Time course analysis of the association of yKu70 protein with DSBs in SLY19 (–Tag) or in its derivatives with <i>YKU70-3FLAG</i> (wild type, MSY4829; <i>tel1</i>Δ, DIY118; <i>xrs2-SH</i>, DIY120). Data are presented as the mean ± SD from four independent trials. Significance was calculated with a Student’s <i>t</i>-test: **<i>p</i> < 0.001; *<i>p</i> < 0.05; n.s., not significant. D. The QAOS method is shown (upper). ssDNA at DSB ends was detected by QAOS assay in wild type (SLY19), xrs2-SH (DIY016), <i>tel1</i>Δ (DIY129) and <i>sae2</i>Δ (DIY059) cells. Data are presented as the mean ± SD from three independent experiments. Significance was calculated with a Student’s <i>t</i>-test: **<i>p</i> < 0.001; *<i>p</i> < 0.05; n.s., not significant. E. The assembly of Tel1 protein on DSBs in SLY19 (–Tag) or in its derivatives with <i>3FLAG-TEL1</i> (wild type, MSY5505; <i>xrs2-314M</i>, MSY5539; <i>xrs2-SH</i>, MSY5486; <i>xrs2-664</i>, MSY5541). F. Association of Sae2 protein with DSBs in SLY19 (–Tag) or in its derivatives with <i>SAE2-3HA</i> (wild type, DIY142; <i>xrs2-314M</i>, DIY144; <i>xrs2-SH</i>, DIY146). In B and F or E, a ChIP assay was performed at the HO cutting site at 150 or 120 min, respectively, after HO induction (DSB +) or before induction (DSB–). Error bars in C, E and F show the SD from three or more independent experiments, each of which consisted of an average from three independent qPCR trials for each strain.</p

The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA–Dependent Tel1 Activation

<div><p>Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.</p></div