Replication Protein-A Mediates the Association of Calf Thymus DNA Polymerase .ALPHA.-DNA Primase Complex with Guanine-Rich DNA Sequence.
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概要
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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 [Suzuki, M. et al. (1993) Biochemistry 32, 12782-12792]. 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.
- 社団法人 日本生化学会の論文
著者
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Yoshida Shonen
Laboratory Of Cancer Cell Biology Research Institute For Disease Mechanism And Control Nagoya Univer
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IZUTA Shunji
Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control. Nagoya Univ
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Savoysky Ericka
Chugai Pharm. Co. Ltd. Fuji Gotemba Res. Labs.
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Suzuki Motoshi
Laboratory Of Cancer Cell Biology Research Institute For Disease Mechanism And Control Nagoya Univer
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Furuta Keigo
Laboratory Of Cancer Cell Biology Research Institute For Disease Mechanism And Control. Nagoya Unive
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Miura Ayuko
Laboratory Of Cancer Cell Biology Research Institute For Disease Mechanism And Control. Nagoya Unive
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Takemura Masaharu
Laboratory Of Biology Education Graduate School Of Mathematics And Science Education Tokyo Universit
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Tamiya-Koizumi Keiko
Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School and Faculty of Medicine
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Furuta Keigo
Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine
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- Replication Protein-A Mediates the Association of Calf Thymus DNA Polymerase .ALPHA.-DNA Primase Complex with Guanine-Rich DNA Sequence.