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Jha, B. ; Ahne, F. ; Eckardt-Schupp, F.

The Use of Double Marker Shuttle Vector to Study DNA Double Strand Break Repair in Wild- Type and Radiation-Sensitive Mutants of the Yeast Saccharomyces Cerevisiae.

Curr. Genet. 23, 402-407 (1993)
DOI
Open Access Green as soon as Postprint is submitted to ZB.
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.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Double-marker plasmid DNA double-strand break Yeast transformation Fidelity of repair
ISSN (print) / ISBN 0172-8083
e-ISSN 1432-0983
Journal Current Genetics
Quellenangaben Volume: 23, Issue: 5, Pages: 402-407 Article Number: , Supplement: ,
Publisher Springer
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Radiation Biology (ISB)