Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb

Bridge-building between mathematical theory and molecular biology: The <em>REV2</em> gene as paradigm.

The DNA damage-repair theory of R.H. Haynes anticipated the possibility of doe-dependent repair processe. The mathematical formalism developed by Haynes and coworkers on the basis of this theory provided tools to probe for the existence of inducible components of mutation or recombination by analysis of dose-response curves. Subsequently, we found that biological and molecular analysis of the Saccharomyces cerevisiae REV2 gene supported the validity of the postulates derived from the mathematical analysis. In this particle, we briefly review the foregoing and summarize ebidence that the REV2 gene product might function in DNA damage-inducible repair and mutation processes

Repair of gamma ray-induced S1 nuclease hypersensitive sites in yeast depends of homologous mitotic recombination and a RAD18-dependent function.

Repair under non-growth conditions of DNA double-strand breaks (DSB) and chromatin sites sensitive to S1 endonuclease (SSS) induced by 60Cobalt-gamma rays were monitored in repair-competent and deficient strains of Saccharomyces cerevisiae by pulsed field gel-electrophoresis. In stationary-phase cells of a repair-competent RAD diploid, and an excision-deficient rad3-2 diploid, SSS are repaired as efficiently as DSB, whereas in a repair-competent RAD haploid, and a rad 50-1 diploid, neither SSS nor DSB are repaired. The rad18-2 diploid repairs DSB well but is defective in SSS repair. Obviously, SSS repair in yeast chromatin, like DSB repair, depends on recombination, but unlike DSB repair depends additionally on RAD18 function

DNA repair genes of <em>Saccharomyces cerevisiae</em>: Complementing <em>rad4</em> and <em>rev2</em> mutations by plasmids which cannot be propagated in <em>Escherichia coli</em>.

The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth