A Versatile Scaffold Contributes to Damage Survival via Sumoylation and Nuclease Interactions

Summary: DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions. : Scaffold proteins are not DNA repair enzymes themselves but make important contributions to DNA repair by regulating and coordinating various enzymes with their DNA substrates. Sarangi et al. reveal the versatility of the Saw1 scaffold by identifying how it copes with several types of DNA damage that depend on its nuclease interactions and sumoylation. These findings highlight the diverse ways in which multifunctional scaffolds can operate under genotoxic stress and how this is directed by protein modification

Sumoylation Influences DNA Break Repair Partly by Increasing the Solubility of a Conserved End Resection Protein

<div><p>Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.</p></div

Sae2 sumoylation site mapping and conserved sumoylation of Sae2 orthologs.

<p><b>A</b>. Sae2 sumoylation is abolished by mutating the Siz SUMO ligases. TAP-tagged Sae2 was immunoprecipitated and its sumoylated form (Sae2-S) was detected as a band migrating above the unmodified band (Sae2) by western blotting with anti-SUMO antibody in wild-type (WT), <i>siz1Δ</i>, and <i>siz2Δ</i> cells, but not in <i>siz1Δ siz2Δ</i> (<i>siz</i>) cells. Unmodified protein was detected by antibody recognizing the TAP tag. <b>B</b>. Sae2 sumoylation is abolished by the <i>K97R</i> mutation <i>in vivo</i>. HA-tagged Sae2 (WT) or Sae2-K97R (KR) expressed from its endogenous promoter was analyzed as in A. <b>C</b>. Sae2-K97R is not sumoylated in <i>E. coli</i>. GST-tagged Sae2 (WT) or Sae2-K97R (KR) co-expressed with SUMO and sumoylation enzymes was examined by western blotting with anti-GST antibody. <i>sae2-K97R</i> abolished the slower migrating form of the protein indicative of lack of sumoylation. <b>D</b>. Schematic of Sae2 showing self-interaction domain, conserved domain and modification sites. Two major Mec1 and Tel1 phosphorylation sites and adjacent site (S249, S278 and T279) are in purple, the S-CDK phosphorylation site (S267) is in green, and sumoylation site (K97) is in red. <b>E–F</b>. The fission yeast and human Sae2 orthologs, SpCtp1 (E) and hCtIP (F) respectively, can be sumoylated in <i>E. coli</i>. (E) HA-tagged SpCtp1 fused with SUMO E2 Hus5 (Hus5-SpCtp1) was co-expressed with SUMO E1 enzymes Rad31 and Fub2 (E1) and GST-tagged fission yeast SUMO (Pmt3) in <i>E. coli</i>. IPTG-induced co-expression of Hus5-SpCtp1 with SUMO and SUMO E1 resulted in the appearance of slow migrating bands above the unmodified Hus5-SpCtp1p on western blots probed with anti-HA antibody, indicative of sumoylation. (F). GST-tagged hCtIP was expressed by IPTG induction with or without the SUMO conjugating enzymes E1 and E2 and SUMO-1 in <i>E. coli</i> as indicated. The soluble protein extracts were analyzed by western blotting using anti-GST antibody.</p

Lack of Sae2 sumoylation impairs Sae2 function.

<p><b>A</b>. Sae2 sumoylation does not affect its protein levels. Sae2-K97R (KR) protein levels are similar to wild-type (WT) before (-) or after treatment with CPT, hydroxyurea (HU) or MMS at 30°C. Extracts from <i>SAE2-TAP</i> cells exposed to the indicated agents were analyzed by western blotting with antibody against TAP. <b>B</b>. Recombination rates at hairpin-capped DSBs. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#s4" target="_blank">Methods</a> for experimental details. Median recombination rates are shown, with the range in brackets. <i>sae2-K97R</i> and <i>sae2Δ</i> exhibit moderate and strong reductions respectively in recombination rates. <b>C</b>. <i>sae2Δ</i> and <i>sae2-K97R</i> show different levels of sensitivity to CPT. 10-fold serial dilutions were used. <b>D</b>. <i>sae2-K97R</i> moderately reduces sporulation efficiency. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#s4" target="_blank">Methods</a> for experimental details. The difference between wild-type and <i>sae2-K97R</i> cells is statistically significant (p<0.05, asterisk). <b>E</b>. <i>sae2-K97R</i> impairs DSB resection in <i>sgs1Δ exo1Δ</i> cells. Left: Schematic illustrating the qPCR-based resection assay. Induction of the HO endonuclease results in a double-strand break at the <i>MAT</i>a locus. The fate of the fragment to the right of the HO cut (dark grey) can be followed by PCR. The unresected, StyI-digested DNA does not yield PCR product using the indicated primer pair (blue arrows), whereas undigested or resected DNA does. Right: The percentage of resected fragment was calculated by the formula detailed in Methods, which compares the PCR yields of digested and mock-digested DNA normalized to amplification at a control locus. For each strain, values from at least three experiments were averaged and standard deviations were calculated. The difference between the two genotypes at indicated time points is statistically significant (p<0.05, asterisk). <b>F</b>. Plasmid-based NHEJ is increased by <i>sae2-K97R</i> in <i>sgs1Δ exo1Δ</i> cells. Cells were transformed with either BamHI-digested or undigested plasmid DNA and plated on media selective for the plasmid. Percentage plasmid repair was calculated by dividing the number of colonies recovered from digested samples with undigested. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#s4" target="_blank">Methods</a> for experimental details. For each genotype, values from at least three experiments were averaged and standard deviations were calculated. Asterisk indicates statistically significant difference (p<0.05). <b>G</b>. <i>sae2-KR</i> suppresses the MMS sensitivity of <i>sgs1Δ exo1Δ</i> cells in a Dnl4-dependent manner. 10-fold serial dilutions were used.</p

Yeast strains used in the study.

<p>Strains in this study are derivatives of W1588-4C, a <i>RAD5</i> derivative of W303 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#pgen.1004899-Zhao2" target="_blank">[88]</a>, unless indicated otherwise. All strains were constructed in this study.</p><p>Yeast strains used in the study.</p

Sumoylation and phosphorylation of Sae2 occur independently and make separate contributions to DNA damage resistance.

<p><b>A</b>. Phosphorylation of Sae2 is unaffected by lack of its sumoylation. Phosphorylation of HA-tagged Sae2 after MMS treatment was examined in the indicated strains. <i>sae2-3A</i> (<i>3A</i>), but not <i>sae2-K97R</i> (<i>KR</i>), abolishes one form of phosphorylated Sae2 (Sae2-P). <b>B–D</b>. Sumoylation level of Sae2 is not affected by lack of Sae2 phosphorylation or the Mec1 kinase. (B). Mutating major Mec1/Tel1 phosphorylation sites does not affect Sae2 sumoylation. (C). Sae2 sumoylation level in Sae2-S267A mutant defective in S-CDK phosphorylation is comparable to wild-type. (D). Deletion of Mec1 does not affect Sae2 sumoylation. Experiments were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#pgen-1004899-g001" target="_blank">Fig. 1A</a>. <b>E–F</b>. Combining mutations of Sae2's sumoylation site and phosphorylation sites results in additivity in CPT and MMS sensitivities. Indicated strains were examined for growth on normal media and media containing either MMS or CPT. 10-fold serial dilutions were used.</p

Sumoylation of Sae2 increases the levels of soluble Sae2.

<p>G1-arrested cells were released into S phase in the presence of 0.03% MMS and cell extracts at indicated time points after release were prepared. Soluble fractions of Sae2-HA from indicated strains were examined by western blots using anti-HA antibody and anti-Adh1 antibody (loading control). Representative results are shown on top, and quantification of the relative amount of soluble Sae2 between the two compared strains from at least three independent trials is shown at the bottom. Asterisks indicate statistically significant differences (p<0.05). <b>A</b>. The level of soluble forms of Sae2 is decreased in <i>sae2-K97R-3A</i> cells compared to <i>sae2-3A</i>. <b>B</b>. The level of soluble forms of Sae2 is decreased in <i>sae2-K97R</i> cells compared to wild-type. <b>C</b>. The level of soluble forms of Sae2 is decreased in <i>sae2-3A</i> cells compared to wild-type.</p

Sumoylation of MRX contributes to DNA end resection.

<p><b>A–B</b>. Sumoylation of the three MRX subunits (Mre11, Rad50 and Xrs2), but not that of Sae2, Lif1, Rad1 or Saw1, is decreased in <i>YKU70-UD</i> cells compared to <i>YKU70-UD*</i>. Experiments were done as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#pgen-1004899-g001" target="_blank">Fig. 1A</a>, and cells were treated with MMS. Triangles indicate the mono-sumoylated forms of the proteins examined. <b>C</b>. <i>YKU70-UD</i>, but not <i>YKU70-UD*</i>, sensitizes <i>sae2Δ</i> to MMS. 10-fold serial dilutions were used. <b>D</b>. Resection is impaired in <i>YKU70-UD</i> compared to <i>YKU70-UD*</i>. qPCR-based resection assay was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#pgen-1004899-g002" target="_blank">Fig. 2E</a>. At least three spore clones for each genotype were tested. Asterisks indicate statistically significant differences (p<0.05). <b>E</b>. <i>sae2-K97R</i> impairs resection in <i>YKU70-UD</i>, but not <i>YKU70-UD*</i>, cells. Assay was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004899#pgen-1004899-g002" target="_blank">Fig. 2E</a>. At least three spore clones for each genotype were tested. Asterisks indicate statistically significant differences between <i>YKU70-UD</i> and <i>YKU70-UD sae2-KR</i> (p<0.05). Note that the values for <i>YKU70-UD</i> and <i>YKU70-UD*</i> are significantly different at all time points (p<0.05). <b>F</b>. Working model for the role of Sae2 sumoylation in DSB resection. MRX denotes the Mre11-Rad50-Xrs2 complex.</p