(A) Individually tagged and immunoprecipitated MCM subunits from yeast strains untreated (−) or treated with 0.3% MMS (+) were western blotted for SUMO (above) and for the relevant protein tags (TAP/Myc, below). Proteins are TAP-tagged unless otherwise indicated. Since a small percentage of protein is typically sumoylated at steady state (see Experimental Procedures), sumoylated forms (upper bands, specifically reduced in the SUMO E2 mutant (mut) background ubc9-1) are only detectable using the SUMO-specific antibody at the exposure shown. The nonspecific region of the SUMO antibody interacts with the Protein A part of TAP, so the lowest band in each lane represents the unmodified form if the protein is TAP-tagged.
(B) Other examples of proteins involved in replication and repair tested for sumoylation. Abf1 (lanes 3–4) and Pob3 (lanes 5–6) are examples of proteins whose sumoylation does not change after MMS.
(C) Spot assay showing 10-fold serial dilutions of yeast strains grown on normal medium (YPD) or with the addition of MMS or HU as indicated.
(D) FACS analysis of cells stained for DNA content (left), and pulsed-field gel stained with ethidium bromide (right), showing a time course of recovery after 1 hr of 0.03% MMS treatment. As., asynchronous cultures. Numbers indicate time post-washout in minutes. The absence of chromosome bands in the gel indicates incomplete replication.

(A) Schematic representation of the chromosome III MAT locus used in the physical assay to assess resection of an HO-induced DSB. 5′–3′ degradation eliminates the StyI (S) and XbaI (X) recognition sites, which results in the disappearance of the StyI/XbaI digestion fragments. Black bars indicate the position of the two probes used for Southern blotting to detect the fragments 0.7kb and 3kb from the break.
(B and C) Examination of DNA end resection and Rad53 phosphorylation after HO cut. Southern blot analysis (top three panels in B) and quantifications (C) show the kinetics of the cut-fragment disappearance. Numbers indicate time post-HO induction in minutes. The bottom panels in (B) are Western blots detecting Rad53 phosphorylation. In (C), the intensity of the cut fragments is plotted relative to that 30 min post-induction. Means from three experiments are presented, error bars indicate SD.
(D–G) Individual proteins from wild-type, tel1Δ, mre11Δ, or sae2Δ strains treated with 0.3% MMS were immunoprecipitated and blotted for SUMO (above) and the protein tag (below) as in Figure 1A. Proteins are TAP-tagged unless otherwise indicated. Untagged strain control (UN) indicates the position of the IgG heavy chain (*), to distinguish it from unmodified Rad59.

(A and B) Individual proteins from wild-type or mec1Δ strains treated with 0.3% or 0.05% MMS were immunoprecipitated and blotted for SUMO (above) and the protein tag (below) as in Figure 1A. Proteins are TAP-tagged unless otherwise indicated.

(A) Protein extracts from asynchronous cultures of sumoylation enzyme mutants untreated (−) or treated with 0.03% MMS for 2 hr (+) were examined for Rad53 (tagged with Flag) phosphorylation (above) and for global sumoylation (middle). Wild-type (WT) and mec1Δ strains are included as respective positive and negative controls for Rad53 phosphorylation. Phospho-Rad53 appears as a band shifted above the unmodified form. The ubc9-10 and adjacent WT cultures (lanes 9–12) were shifted to 37°C for 30 min before harvesting. Protein stain (amido black, below) indicates loading.
(B) Rad53 phosphorylation was examined following G1 culture (0) release into 0.03% MMS for the indicated number of minutes. FACS analysis of DNA content from the same samples is shown (right). siz1Δ siz2Δ and siz1Δ mms21-CH mutants and adjacent wild-type strains were cultured at 30°C and Flag-tagged Rad53 was detected by anti-Flag antibody (top two blots). The time course for the ubc9-10 mutant and adjacent wild-type control strain was carried out at 37°C, and untagged Rad53 was detected using a Rad53-specific antibody (lower blot).
(C) Immunoprecipitated checkpoint signaling proteins from yeast strains untreated (−) or treated with 0.3% MMS (+) were western blotted for SUMO (above) and for the relevant protein tags (TAP/Myc/Flag, below) as in Figure 1A. Phosphorylation of Dun1 and Rad53 appears as a band shift. Sumoylated Dun1 is detectable as an additional faint band above the shifted form. The lower band under Rad53 is a nonspecific band. Proteins are TAP-tagged unless otherwise indicated.
(D) Spot assay showing ten-fold serial dilutions of yeast strains grown on normal medium (YPD) or with the addition of MMS or HU as indicated.
(E) Simplified scheme showing how sumoylation may be integrated within the overall DNA damage response. DNA damage-induced sumoylation is abbreviated as “SUMO response.” “Phos” indicates phosphorylated.