Home › Cell Biology › Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae
Cell Biology JoVE (Open Access) Citable · DOI

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

DOI: 10.3791/3150-v
What you'll learn
  • ✓ Quantify HO-stimulated translocation frequencies in diploid yeast strains
  • ✓ Perform Southern blot and chromosome blot analysis of DNA rearrangements
  • ✓ Interpret single-strand annealing mechanisms in double-strand break repair
  • ✓ Model ionizing radiation-induced genome rearrangements using yeast assays
Protocol

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Difficulty
advanced
Total time
~5–7 days (includes strain growth, HO induction, plating, Southern/chromosome blot hybridization and analysis)
Model organism
Saccharomyces cerevisiae (diploid strain)
Biosafety
BSL-1

Steps

1
Determine HO-stimulated translocation frequencies

Induce HO endonuclease expression in diploid yeast to create double-strand breaks at multiple loci, then measure translocation events by colony survival and genetic analysis.

▶ 01:16
2
Determine plating efficiencies

Calculate plating efficiency of control and HO-induced strains to normalize translocation frequency data and account for differences in cell viability.

▶ 02:40
3
Perform Southern blot analysis

Extract genomic DNA, digest with restriction enzymes, separate by gel electrophoresis, and hybridize with labeled probes to detect rearranged loci.

▶ 04:05
4
Perform chromosome blot analysis

Separate intact chromosomes by pulsed-field gel electrophoresis and hybridize with probes to visualize translocated chromosome structures genome-wide.

▶ 04:57
5
Quantitate and analyze chromosomal translocations

Measure band intensity and size from Southern and chromosome blots to quantify translocation frequency, characterize rearrangement junctions, and correlate with single-strand annealing mechanisms.

▶ 06:13
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