HYPERMUTABLE LIGATION OF PLASMID DNA ENDS IN CELLS FROM PATIENTS WITHWERNER SYNDROME

Werner Syndrome is a rare autosomal recessive disorder characterized b y an increased cancer risk and by symptoms suggestive of premature agi ng. Cells from these patients demonstrate a typical pattern of chromos omal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied the in vivo DNA ligation i n three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected li nearized plasmid pZ189 and measured the amount of plasmid DNA ends rej oined by these host cells as the ability of the recovered plasmid to t ransform bacteria. A mutagenesis marker gene close to the ligation sit e allowed screening for mutations. Subsequent mutation analysis provid ed information about the accuracy of the ligation process. The cells f rom Werner syndrome patients were as effective as normal cells in liga ting DNA ends. However, mutation analysis revealed that the three Wern er syndrome cell lines introduced 2.4-4.6 times more mutations (p < 0. 001) than the normal cell lines during ligation of the DNA ends: the m utation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, a nd 24.4% in the normal cell lines. These increased mutation frequencie s in plasmids ligated during passage through Werner syndrome cells wer e mainly due to a significant (p < 0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hype rmutability and the genomic instability in Werner syndrome cells and r elated to the apparently accelerated aging and high cancer risk in aff ected patients.