Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template.
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.
Braguglia
D
D
Swiss Institute for Experimental Cancer Research, Ch. des Boveresses 155, Epalinges/Lausanne, CH-1066, Switzerland.
Heun
P
P
Pasero
P
P
Duncker
B P
BP
Gasser
S M
SM
D000251:Adenosine Triphosphatases / Q000502:physiology*; D016590:Aphidicolin / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004265:DNA Helicases; D019175:DNA Methylation; D004256:DNA Polymerase I / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004278:DNA, Superhelical / Q000378:metabolism* / Q000648:ultrastructure; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics / Q000648:ultrastructure; D012313:RNA / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004278:DNA, Superhelical; D003838:Deoxyadenine Nucleotides; D003856:Deoxyuracil Nucleotides; D009687:Nuclear Proteins; C032549:bromodeoxyuridine triphosphate; D016590:Aphidicolin; D012313:RNA; D004256:DNA Polymerase I; D004258:DNA Polymerase III; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C026600:2'-deoxyadenosine triphosphate