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 pha se. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1). In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating s ystem. Semiconservative DNA replication is monitored by substitution of new ly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and dens ity gradient analysis. In addition, neutral-neutral two-dimensional gel ana lyses and, in the case of nuclei, detection of newly synthesized DNA in rep lication foci by DIG-dUTP incorporation can be used to monitor replication. (C) 1999 Academic Press.