title: Assessment of yeast chromosome XII instability: single chromosome comet assay.
abstract: The tools and techniques used in single-cell analysis of DNA damage in yeast Saccharomyces cerevisiae are limited. In this study, we modified the single cell gel electrophoresis assay, namely, the single chromosome comet assay based on DNA break analysis, at the chromosomal level. We studied the largest yeast chromosome XII, which contains the rDNA locus, and we investigated its instability using cell cycle checkpoint-, DNA damage- and antioxidative defence-deficient, and lifespan-deregulated yeast mutant strains. Moreover, we compared chromosome XII instability with the variability of nucleolar rDNA fluorescence signals. Three single-gene-deletion strains, cells lacking single-stranded DNA endonuclease, Rad1p; NAD(+)-dependent histone deacetylase, Sir2p; and gamma glutamylcysteine synthetase, Gsh1p, were more prone to chromosome XII instability compared to corresponding wildtype strains, indicating that DNA damage repair machinery, chromatin silencing and redox homeostasis may contribute to genome stability. Elevation in the number of DNA breaks was correlated with a high variability in the levels of nucleolar rDNA in the Δrad1 background, while unaffected chromosome XII and low variability in nucleolar rDNA fluorescence signals were observed in the Δtor1 longevity mutant. Taken together, the single chromosome comet assay may be successfully used to study DNA damage at the chromosomal level, which might be overlooked using whole population analysis on DNA breaks with PFGE separation.
authors: Department of Biochemistry and Cell Biology, University of Rzeszow, Poland; Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Kolbuszowa, Poland.
authors: Anna
authors: A
authors: Lewinska
authors: Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Kolbuszowa, Poland; Department of Genetics, University of Rzeszow, Rejtana 16C, PL 35-959 Rzeszow, Poland.
authors: Beata
authors: B
authors: Miedziak
authors: Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Kolbuszowa, Poland; Department of Genetics, University of Rzeszow, Rejtana 16C, PL 35-959 Rzeszow, Poland. Electronic address: mawnuk@gmail.com.
authors: Maciej
authors: M
authors: Wnuk
mesh_terms: D043171:Chromosomal Instability; D015825:Chromosomes, Fungal / Q000235:genetics*; D020552:Comet Assay; D004249:DNA Damage; D004260:DNA Repair; D004275:DNA, Ribosomal / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D020868:Gene Silencing; D042822:Genomic Instability; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D004275:DNA, Ribosomal
keywords: DNA damage; Fluorescence in situ hybridisation; Single chromosome comet assay; Whole chromosome painting probe; Yeast; rDNA locus