SEMICONSERVATIVE REPLICATION IN YEAST NUCLEAR EXTRACTS REQUIRES DNA2 HELICASE AND SUPERCOILED TEMPLATE

We describe the preparation of nuclear extracts from yeast cells synch ronised In S-phase that support the aphidicolin-sensitive, semi-conser vative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replica tion intermediates that have incorporated [alpha-(32)]dATP, by the con version of methylated template DNA into a hemi-methylated or DpnI-resi stant form, and by substitution of dTTP with the heavy derivative BrdU TP, which results in a shift in density corresponding to complete seco nd strand synthesis. We demonstrate dependence on DNA pol delta and th e pol alpha/primase complex, and are able to detect putative Okazaki f ragments under ATP-limiting conditions. In contrast to the semi-conser vative replication of supercoiled plasmid, linear or open-circular tem plates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent wi th a true replication reaction we find that semi-conservative replicat ion of plasmid DNA is stimulated in S-phase relative to G(1)-phase nuc lear extracts, and is independent of the recombination-promoting facto r Rad52p. Using this novel system we demonstrate that semi-conservativ e replication, but not polymerase activity per se, requires the activi ty of the DNA helicase encoded by DNA2. (C) 1998 Academic Press.