Journal Article

The Carboxyl Terminal Sequence of Nucleolar Protein B23.1 Is Important in Its DNA Polymerase α-Stimulatory Activity

Hayato Umekawa, Keiji Sato, Masaharu Takemura, Yoshinori Watanabe, Satomi Usui, Takao Takahashi, Shonen Yoshida, Mark O.J. Olson and Yukio Furuichi

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 130, issue 2, pages 199-205
Published in print August 2001 | ISSN: 0021-924X
Published online August 2001 | e-ISSN: 1756-2651 | DOI:
The Carboxyl Terminal Sequence of Nucleolar Protein B23.1 Is Important in Its DNA Polymerase α-Stimulatory Activity

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The protein B23 is a major nucleolar phosphoprotein comprising two isoforms, B23.1 and B23.2, which differ only in their carboxyl-terminal short sequences, the N-terminal 255 residues being identical in both forms. Both B23.1 and B23.2 stimulated immunoaf-finity-purified calf thymus DNA polymerase α in a dose-dependent manner. The stimulatory effect of protein B23.1, the longer isoform, was found to be 2-fold greater than that of B23.2. Purified DNA polymerase a bound tightly to a protein B23.1-immobilized column, while it bound weakly to a protein B23.2-immobilized column. Surface plasmon resonance studies by BIAcore further showed that protein B23.1 bound to the DNA polymerase α-(dA)-(dT) complex more tightly than did protein B23.2. The protein B23 isoforms appear to interact directly with the DNA polymerase α protein and not through the bound nucleic acid. These observations indicated that protein B23 physically bound to the DNA polymerase α and stimulated the enzyme activity. Product analyses showed that protein B23 greatly enhanced the reaction both in amount and length of product DNA, whereas it did not significantly alter the processivity of polymerization. In contrast, protein B23 effectively protected DNA polymerase α from heat inactivation. These results suggest that protein B23 stabilizes DNA polymerase α that is detached from product DNA, allowing the enzyme to be recruited for further elongation. Moreover, experiments using various C-terminal deletion mutants of protein B23 indicated that 12 amino acids at the C-terminal end of B23.1, which are absent in B23.2, may be essential for the full stimulation of the DNA polymerase α.

Keywords: DNA polymerase α; protein B23; protein isoforms

Journal Article.  0 words. 

Subjects: Biochemistry

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