Article: article from journal or magazin.
Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template.
Journal of Molecular Biology
We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [alpha-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2.
Adenosine Triphosphatases/physiology, Aphidicolin/pharmacology, Cell Cycle/physiology, Cell Nucleus/metabolism, Centrifugation, Density Gradient, DNA Helicases, DNA Methylation, DNA Polymerase I/metabolism, DNA Polymerase III/metabolism, DNA Replication/genetics, DNA, Superhelical/metabolism, DNA, Superhelical/ultrastructure, Deoxyadenine Nucleotides/metabolism, Deoxyribonucleases, Type II Site-Specific/metabolism, Deoxyuracil Nucleotides/metabolism, Electrophoresis, Gel, Two-Dimensional, Microscopy, Electron, Nuclear Proteins/metabolism, Nucleic Acid Conformation, Plasmids/genetics, Plasmids/ultrastructure, RNA/metabolism, Saccharomyces cerevisiae/enzymology
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