Replication protein-A mediates the association of calf thymus DNA polymerase α-DNA primase complex with guanine-rich DNA sequence

Motoshi Suzuki, Keiko Tamiya-Koizumi, Masaharu Takemura, Keigo Furuta, Shunji Izuta, Ericka Savoysky, Ayuko Miura, Shonen Yoshida

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Abstract

We have shown that calf thymus DNA polymerase α-DNA primase complex (polα-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis. Here we tested the association of polα-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication. While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with polα-primase using anti-polα antibody, SVG was hardly precipitated under the same conditions. Competition studies in either gel-retardation assay or during de novo DNA synthesis by polα-primase demonstrated that the interaction of polα-primase with SVG was much weaker than that with CTPPS 1. On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1. After preincubation with RP-A, SVG could bind polα-primase that was immobilized on Sepharose beads. The simian virus 40 large T antigen also enhanced association of SVG to polα-primase, while Escherichia coli single-stranded DNA-binding protein did not. However, polα-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers. When SVG was extended 10 nucleotides at its 5'-end, polα-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A. These results suggest a new role for RP-A, i.e., as a molecular tether that allows polα-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.

Original languageEnglish
Pages (from-to)766-772
Number of pages7
JournalJournal of Biochemistry
Volume120
Issue number4
DOIs
Publication statusPublished - 01-01-1996
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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