<em>In vitro</em> monitoring of base excision repair in <em>Saccharomyces cerevisiae</em>.

Base excision repair (BER) is an important mechanism to maintain genomic stability. Here we offer a set of protocols to quantitatively analyze BER capacity in whole cell-free yeast extracts. Cell-free yeast extracts were obtained by a French press procedure and repair capacities were measured by using oligonucleotide substrates. Repair products were separated by polyacrylamide gel electrophoresis and detected by autoradiography. These set of methods allow the analysis of different kinds of base damage and of individual mechanistic steps within BER. We used these protocols to investigate a new role of the DNA double strand break repair protein XRS1 in BER (1)

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

Molecular analysis of the REV 2 gene of <em>Saccharomyces cerevisiae</em> - a review.

The REV2 gene controls DNA repair, induced mutagenesis and, probably, some fidelity mechanism of replication. Of particular interest is the notion that it is inducible by DNA-damaging agents. We wanted to find molecular evidence for these results derived from numerous biological experiments. We cloned the REV2 gene from a yeast genomic DNA library based on the YCp 50 centromere vector, sequenced it and studied its regulation on the transcriptional level. The coding region of the REV2 gene consists of a 1425 pb reading frame with a coding capacity for a polypeptide of 52 kD; no significant homology to any gene filed in available data bases was found. Examination of a hydrophobicity plot of the putative Rev2 protein predicts the existence of transmembrane helices. Quantitative Northern analysis confirmed the working hypothesis that DNA-damaging agents increase the level of REV2 gene expression in stationary cells. Thus, the REV2 gene seems to code for a membrane protein which is inducible by DNA-damaging agents and which controls processes of repair and mutagenesis in yeast

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast <em>Saccharomyces cerevisiae</em>.

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90 % in RAD cells (normalized for the expression of undamaged URA3 in TRP+ transformants). Plasmids isolated from the transformants (URA+ TRP+) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20 %) for the correct repair of both DSBs and DSGs