In Vitro DNA Replication in Yeast Nuclear Extracts

Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G1. In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system. Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5ʹ-triphosphate (BrdUTP) and density gradient analysis. In addition, neutral–neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication.