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Fig. 4. ssDNA is enriched in Myc-Mec1 precipitates. (A) Schematic for QAOS (Booth et al., 2001) near the HO cleavage site of MATalpha is shown. (B, left) The percentage of HO1 signal that exists as ssDNA was measured by QAOS in the input DNA at the indicated time points after HO cut induction. (Right) Myc-Mec1 ChIP was performed in JKM179 after galactose induction of the HO endonuclease. Fold enrichment of Mec1 over SMC2 at the HO2 site (0.6 kb away from the HO cleavage site) is shown. (C) The efficiency of HO cut-site cleavage (Fig. 1A, shown here in blue) is plotted against the percentage of ssDNA at HO1 in input DNA (Fig. 2B, red), the percentage of cells showing a single bright Mec1 focus (Fig. 1B, green) and the fold enrichment of Myc-Mec1 at the DSB over SMC2 (Fig. 1C, HO2, black). (D) Myc-Mec1 ChIP was performed after galactose induction of the HO endonuclease as in Fig. 1C. The IP DNA was then subjected to QAOS to quantify the fraction of the precipitated HO1 site that is ssDNA. The amount of ssDNA (light grey) is plotted as a fraction of the total Myc-Mec1-bound DNA. (E) Schematic of the degradation of Myc-Mec1-bound DNA by the RecJ exonuclease. RecJ is a 5'-to-3' exonuclease that degrades only ssDNA. It will not degrade DNA that is ds at the 5' end. (F) The amount of ssDNA at HO1 was measured by QAOS on Myc-Mec1-bound DNA at the indicated time points after HO induction either before or after incubation with RecJ endonuclease for 3 hours at 37°C, but after removal of proteins by proteolysis and phenol extraction.
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