Analysis of telomeric DNA replication using neutral-alkaline two-dimensional gel electrophoresis

Abstract

DNA replication studies often rely on analysis of replication intermediates, such as progressing replication forks and growing nascent strands. The assay presented here for replication at telomeres in the yeast Saccharomyces cerevisiae is based on the analysis of nascent DNA strands prior to the ligation step. Preligation replication intermediates are very rare due to their transient nature. To enrich for such intermediates, inhibition of the ligation step is performed by using a temperature-sensitive allele of the replicative ligase Cdc9 at nonpermissive temperature. The method can be used for fine analysis within rather short DNA fragments which makes it particularly advantageous for studying telomere replication. It can also be helpful for analysis of DNA recombination and potentially any process which involves ligation of nicked DNA. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.