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.