Nromalized For Mat Dsb Efficiency Ho

For mat switching genomic dna was digested with styi and bglii fig.

Nromalized for mat dsb efficiency ho.

We showed that the repair efficiency of phleomycin induced dsb in elg1 mutants was lower than wild type cells in g 2 m phase figure 2. The efficiency of dsb formation at hocs is measured with the olwdh1766 and olwdh1767 primers. It is normalized to the uncut levels i e prior to galactose addition. For allelic recombination dna was digested with bglii and pvui and probed with the mat distal probe to check the efficiency of ho induction and with a mat proximal probe to check the appearance of the product.

Dna double strand breaks dsbs are repaired by two principal mechanisms. Download full size image. A relative fold enrichment of ino80 myc chip for wt and hta1 2 s129 strains at the indicated distances from the ho cut site and the indicated times on galactose the enrichment is corrected for cleavage efficiencies which were 96 98 and 99 wt and 89 98 99 hta1 2 s129. A single double strand break dsb induced by ho endonuclease triggers both repair by homologous recombination and activation of the mec1 dependent dna damage checkpoint in budding yeast1 2 3 4 5 6.

The kinetics and frequency of crossover outcomes in mitotic cells were studied in an interchromosomal recombination system a dsb within a 2 kb mata sequence inserted in chromosome v is created by a galactose inducible ho endonuclease. 2b d and probed with a mat distal probe. Non homologous end joining nhej and homologous recombination hr 1 hr is the most accurate dsb repair mechanism but is generally restricted to the s and g2 phases of the cell cycle when dna has been replicated and a sister chromatid is available as a repair template 2 5 by contrast nhej operates throughout the cell. In the ho endonuclease induced recombination system elg1 associated with both sides of the ho induced dsb site figure 4 suggesting that elg1 plays a role in early events in hr or the damage response.

Special attention has been paid to the switching of mating type genes in saccharomyces cerevisiae a process initiated by the ho endonuclease by cleaving the mat locus. The ability to induce synchronously a single site specific double strand break dsb in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during dsb repair. Download high res image 254kb download. The results obtained by this method are not different from southern blot quantification fig.

The plating efficiency pe of mph1δ cells on medium containing galactose constitutive ho expression relative to growth on glucose containing medium was 78 not significantly different from wild type p 0 59 by contrast the pe of the sgs1δ mutant was significantly reduced p 0 034 consistent with a previous study ira et al 2003. A relative fold enrichment of ino80 myc chip for wt and hta1 2 s129 strains at the indicated distances from the ho cut site and the indicated times on galactose the enrichment is corrected for cleavage efficiencies which were 96 98 and 99 wt and 89 98 99 hta1 2 s129. 2a or with styi fig.