JCS018366 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Gel electrophoresis of the PCR products of Myc-Mec1 ChIPs for wild-type, yku70, ddc2 sml1 and rad9 rad24 strains as described in Figs 1, 2 and 4, respectively. The HO endonuclease was induced by growth on galactose for 0.5, 1, 2 or 4 hours. The 0-hour time point represents cells exposed to glucose for 2 hours to repress HO expression. Protein-DNA complexes were immunoprecipitated (IP) using anti-Myc (9E10) or anti-HA (12CA5) as a negative control. DNA purified from INPUT (IN), the Myc-Mec1 (Myc) or HA (HA) IPs was analysed by multiplex PCR primers for the three HO sites and one pair within the uncleaved SMC2 gene as described in Fig. 1. A representative gel of the PCR products is shown. Quantitation of the products in A-D is presented in Figures 1, 2 and 4 as the ratio of the HO1, HO2 or HO3 signals to SMC2 in the IP, normalized to the same ratio in the corresponding INPUT.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. (A) ChIP for Myc-Mec1 at the indicated time points after induction of GAL::HO endonuclease on galactose performed in strain GA1529. The 0-hour time point represents cells exposed to glucose for 2 hours to repress HO expression. Anti-Myc (9E10) was used for ChIP. DNA purified from INPUT (IN) and Myc-Mec1 (Myc) was analysed by rtPCR primers for sites at indicated distances from the HO cleavage consensus. Absolute fold enrichment was calculated as follows: for each time point, the signal from a site near the HO DSB at MAT was normalized to that from the noncleaved SMC2 locus in ChIP and input DNA samples. For each time point and site, the normalized ChIP signals were normalized to input DNA signals, because end-resection can reduce the available DNA template.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Mec1 foci form in tel1 deficient strains following DSB induction. Myc-Mec1 immunofluorescence was performed as described in Fig. 1B except that the JKM179 derivative carried a complete deletion of tel1. Mab414 labels nuclear pores (red) and anti-Myc is in green on cells treated either with 2% glucose (0 h) or after 6-hours of incubation in galactose. Arrows indicate cells with large single Mec1 foci.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. The ssDNA at HO1 was confirmed by performing QAOS on DNA immunoprecipitated with Mec1 after 4 hours of HO induction, with or without incubation with the Mung-bean nuclease for 3 hours at 37°C. Mung-bean-nuclease digestion is performed after the removal of proteins by proteolysis and phenol extraction. As control, a dsDNA fragment and total DNA (Input) from the ChIP isolated after 4 hours on galactose was treated or not with the Mung-bean nuclease and quantified for ssDNA. Moreover, an internal fragment from the SMC2 gene was amplified by PCR to control for the presence and integrity of DNA the ChIP samples.

• Supplemental Table S1 - Adobe PDF