Cell survival after MMS damage. (A) Sensitivity of yeast strains treated with 11.8 mM (0.1%) MMS for 15 or 30 min in PBS and plated, after 24 h of LH repair, on rich medium from serial 10-fold dilutions. (B) Survival of MMS-treated G₁ yeast (after 24 h of LH). ○, WT; □, rad27Δ mutant; •, pol32Δ mutant; ▴, rad27-p mutant; ▪, pol32Δ rad27-p mutant.

Determination of SSBs by S1 nuclease treatment. MMS treatment of chromosomal DNA of WT yeast within plugs, along with the presence or absence of S1 nuclease, is indicated. DNA plugs were incubated in buffer either at 4°C (no heat treatment; lanes 1 to 10) or at 55°C (heat treatment) for 24 h to convert heat-labile sites into SSBs (lanes 11 to 20). Thereafter, S1 nuclease (4 U/ml for 20 min) treatment was applied to both mock heat-treated (lanes 6 to 10) and heat-treated (lanes 16 to 20) plugs.

Comparable repair of total MMS-induced lesions in WT and pol32Δ rad27-p strains. Genomic DNAs were isolated from mock-treated and MMS-treated (15 and 30 min) G₁ haploid cells before and after LH. QPCR was performed to amplify a 10-kb fragment and compared with controls to calculate the relative amplification. Estimation of the average number of lesions was done as described by Ayala-Torres et al. (1). Each bar represents the mean value and standard error for four independent MMS treatments (including six independent QPCR analyses for each sample).

Accumulation of DSBs in pol32Δ rad27-p double mutants. G₁ haploid yeast cells of different genotypes were treated with 0.1% (11.8 mM) MMS for 15 min (lanes 2, 5, 8, 11, 14, 17, 20, and 23) or 30 min (lanes 3, 6, 9, 12, 15, 18, 21, and 24), followed by immediate DNA purification and PFGE (lanes 1 to 12) or PFGE after LH for 24 h (lanes 13 to 24). Lanes 1, 4, 7, 10, 13, 16, 19, and 22 are mock-treated controls. Chromosomal DNA preparation with proteinase K digestion was performed either at 30°C (A) to measure preexisting closely opposed SSBs formed in vivo or at 55°C (B) (the order of lanes is the same as in panel A) to measure total closely opposed lesions. Chromosomes were visualized by ethidium bromide staining. Positions of some chromosome bands are indicated. A broken chromosome III that can be detected by Southern blotting is located between Chr I and Chr IX. The smear below the Chr I band (indicated by an asterisk) is nonspecific DNA material. A Southern blot detecting the two chromosome bands (Chr II and Chr III) is shown below the agarose gel (30°C).

A mag1 deletion in the pol32Δ rad27-p background prevents accumulation of DSBs. Genotypes are indicated. Each group of three lanes (from left to right) contains a mock-treated control and samples with 15 min and 30 min of MMS treatment. (A) DNA samples were processed at 30°C to detect closely opposed SSBs that were formed in vivo. (B) DNA samples were processed at 55°C to detect total closely opposed lesions (methylated lesions, AP sites, and SSBs). A Southern blot and the number of DSBs per haploid yeast genome, calculated as described in Materials and Methods, are shown under each lane.

SSBs induced by MMS treatment are repairable in pol32Δ rad27-p mutants. Each group of three lanes (from left to right) represents WT, pol32Δ rad27-p, and rad27 genotypes. G₁ haploid yeasts were treated with MMS as follows: mock-treated control (lanes 1 to 3 and 10 to 12), 15 min of MMS treatment (lanes 4 to 6 and 13 to 15), and 15 min of MMS treatment followed by LH (lanes 7 to 9 and 16 to 18). Chromosomal DNA samples were processed at 30°C in order to detect SSBs formed in vivo. S1 nuclease treatment was applied to DNA plugs (lanes 10 to 18).

Possible mechanisms for DSB formation from closely spaced lesions. When there is coordination of BER enzymes (WT), closely opposed base lesions can be repaired one by one. Once the first lesion is processed by glycosylases followed by AP endonucleases, strand displacement and 5′-flap removal are quickly finished to seal the nick generated during repair before starting nicking of the second lesion, thus avoiding the formation of DSBs. When there is a defect in the coordination of BER enzymes (pol32 rad27-p mutant), the strand displacement and/or 5′-dRP end flap removal might be slowed, resulting in increased time and space for the generation of a second nick 5′ of the closely opposed lesion, thereby leading to DSB formation. In this case, widely separated intermediate SSBs (e.g., >30 nt apart) might, in essence, be brought together to form DSBs. Alkylated bases are shown as filled red circles. 5′-dRP is shown as filled blue circles. Arrowheads on DNA strands correspond to 3′ ends. Red dashed lines represent repair-associated DNA synthesis, and red solid lines represent newly synthesized DNA. Note that this figure shows only one orientation of the two closely opposed SSBs, where DNA synthesis and strand displacement intermediates face each other. DSBs would not be generated if DNA synthesis and strand displacement at closely opposed SSBs proceeded in the opposite orientation.