Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase α and δ during initiation of leading and lagging strand synthesis

Toshiki Tsurimoto, B. Stillman

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Abstract

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases α and δ. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase α activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases α and δ. The primer recognition complex, RF-C·PCNA·ATP, stimulated pol δ activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase α. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase α dependent synthesis of lagging strands and DNA polymerase δ/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto,T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.

Original languageEnglish
Pages (from-to)1961-1968
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number3
Publication statusPublished - 1991
Externally publishedYes

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DNA-Directed DNA Polymerase
DNA Replication
DNA
Proliferating Cell Nuclear Antigen
Replication Protein C
Replication Protein A
Single-Stranded DNA
Plasmids
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase α and δ during initiation of leading and lagging strand synthesis",
abstract = "Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases α and δ. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase α activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases α and δ. The primer recognition complex, RF-C·PCNA·ATP, stimulated pol δ activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase α. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase α dependent synthesis of lagging strands and DNA polymerase δ/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto,T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.",
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AU - Stillman, B.

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Y1 - 1991

N2 - Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases α and δ. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase α activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases α and δ. The primer recognition complex, RF-C·PCNA·ATP, stimulated pol δ activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase α. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase α dependent synthesis of lagging strands and DNA polymerase δ/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto,T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.

AB - Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases α and δ. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase α activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases α and δ. The primer recognition complex, RF-C·PCNA·ATP, stimulated pol δ activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase α. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase α dependent synthesis of lagging strands and DNA polymerase δ/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto,T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.

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