Analysis of DNA double strand breakage and repair using orthogonal field alternation gel electrophoresis.

Orthogonal field alternation gel electrophoresis (OFAGE) allows separation of DNA molecules in the size range of 200 kb to 3000 kb. These sizes encompass the chromosome sizes of the genome of Saccharomyces cerevisiae. Using this technique, we have found that yeast cells exposed to X-rays generate a smear of DNA fragments corresponding to the products of random, independent double strand breaks, and that the bands corresponding to unbroken chromosomes decrease in intensity in direct proportion to chromosome size. If exposed wild type cells are permitted time to repair (5 h at 30 degrees C on YEPD), the fragments partially disappear and the chromosome bands reappear, although at less than normal intensity. In certain radiation-sensitive mutants (rad51, rad52 and rad54), the fragment smear appears following X-ray exposure but no repair of broken chromosomes occurs. In fact, loss of the fragments occurs; this could appear as partial repair using other procedures.