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.