Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen.
Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.
Mitkova
Atanaska V
AV
Department of Molecular Biology, School of Osteopathic Medicine & Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
Biswas
Esther E
EE
Biswas
Subhasis B
SB
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002411:Cation Exchange Resins; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives*; D002852:Chromatography, Ion Exchange; D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics; D006131:Growth Inhibitors / Q000494:pharmacology; D007106:Immune Sera / Q000494:pharmacology; D051379:Mice; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D010957:Plasmids / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000378:metabolism*; D051716:Replication Protein A; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*
D002411:Cation Exchange Resins; D004271:DNA, Fungal; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006131:Growth Inhibitors; D007106:Immune Sera; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; D016590:Aphidicolin; D002482:Cellulose; C008881:phosphocellulose; D004259:DNA-Directed DNA Polymerase