Abstract
DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.
Highlights
Andrew Murray and Jack Szostak [1] constructed the first Yeast Artificial Chromosome (YAC) soonafter Szostak and Blackburn succeeded to clone yeast telomeres on linear plasmid vectors [2]
It was immediately obvious that the experiment had worked, and that the Tetrahymena ends were able to act as functional telomeres in yeast’’ [4]. ¿Why does small linear YACs appear as a single band when separated by gel electrophoresis? - Probably because they do not support supercoiling
While many bands with different electrophoretic mobility were detected for the circular pYAC_MEM isolated from S. cerevisiae in lane 1, no topoisomers were observed for the intact form of the linear YAC_MEM isolated from S. cerevisiae in lane 3
Summary
Andrew Murray and Jack Szostak [1] constructed the first Yeast Artificial Chromosome (YAC) soonafter Szostak and Blackburn succeeded to clone yeast telomeres on linear plasmid vectors [2] This was the beginning of a crucial series of experiments that led to a whole new field: Molecular Cytogenetics. It was early recognized that small YACs are less stable than natural yeast chromosomes and buildup stability as their size increases [3], they do replicate and segregate In his Nobel Prize Lecture, Szostak describes the revealing observation that indicated he succeeded to obtain the first YAC: ‘‘When DNA molecules are separated by gel electrophoresis, circles generate a series of bands corresponding to monomers and multimers, and relaxed and supercoiled forms, leading to a complicated pattern. The results obtained indicated that Topo 2 is dispensable for the replication and segregation of small linear YACs
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