Use of Pulsed-Field Gel Electrophoresis for Studies of DNA Double-Strand Break Repair in the Yeast Saccharomyces cerevisiae

Abstract

We present a sensitive, nonradioactive method for DNA double-strand break (DSB) quantification in the yeast Saccharomyces cerevisiae (or any other organism with linear chromosomes ≤9 Mb). Our technique allows studies of induction, repair, and local distribution of DSB induced by radiation and chemicals or of physiological origin. DSBs cause a loss of intact chromosomal length DNA and a simultaneous increase in DNA fragments. We use pulsed-field gel electrophoresis to separate the DNA molecules; stain the gels with ethidium bromide, which yields a fluorescence signal proportional to DNA mass; and monitor the fluorescence intensity distribution along the gel lanes with a charge-coupled device camera-based image analysis system. To evaluate the DNA mass profiles, the computer program PULSE was established. It calculates predicted DNA mass distributions, which depend on an adequate model for the spatial distribution of DSB and on arbitrary values for the DSB frequency per unit length, α. Furthermore, the migration behavior of DNA molecules in the gels is accounted for by an empirical calibration of migration distances vs molecular length and an adequate model for the gel response function. The actual breakage frequency, α 0, is finally obtained by determining and minimizing deviations between calculated and observed profiles, where α 0 is the parameter value yielding the best fit. With sparsely ionizing radiation and after repair incubation it turned out that a so-called random breakage model with a constant probability for breakage at a specific site is most suitable for describing the observed DNA mass distribution in yeast chromatin.