Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex.
We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.
Mitkova
Atanaska V
AV
Department of Molecular Biology, GSBS & SOM, University of Medicine & Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
Biswas-Fiss
Esther E
EE
Biswas
Subhasis B
SB
D002474:Cell-Free System; D004256:DNA Polymerase I / Q000502:physiology; D004258:DNA Polymerase III / Q000502:physiology; D004261:DNA Replication; D026942:DNA Topoisomerases / Q000502:physiology; D004264:DNA Topoisomerases, Type I / Q000502:physiology; D004278:DNA, Superhelical / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D026341:Minichromosome Maintenance 1 Protein / Q000502:physiology; D051738:Origin Recognition Complex; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology
C061886:ABF1 protein, S cerevisiae; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D026341:Minichromosome Maintenance 1 Protein; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D026942:DNA Topoisomerases; D004264:DNA Topoisomerases, Type I