Amino acid sequence motifs essential to 3' → 5' exonuclease activity of Escherichia coli DNA polymerase II

Y. Ishino, H. Iwasaki, I. Kato, H. Shinagawa

研究成果: ジャーナルへの寄稿記事

19 引用 (Scopus)

抄録

Many DNA polymerases have conserved sequences required for 3' → 5' exonuclease activity, which contributes to the accuracy of DNA replication by removing misincorporated nucleotides prior to chain elongation. Using amino acid sequence alignments, we predicted the putative active site of the 3' → 5' exonuclease of Escherichia coli DNA polymerase II. Site-directed mutagenesis at D155A, E157A, D155A/E157A, D228A, Y330F, and D334A, which are in the predicted exonuclease active regions, specifically inactivated 3' → 5' exonucleolytic activity but not DNA-polymerizing activity of E. coli DNA polymerase II. Furthermore, all of the mutants were diminished in the in vitro proofreading ability, as judged by their increased insertion and extension of wrong nucleotides. These findings indicate that the 3' → 5' exonuclease region of the E. coli DNA polymerase II is in the amino-terminal part of the protein, as it is in other DNA polymerases, and are consistent with the proposal of an evolutionary conserved 3' → 5' exonuclease active site in most DNA-dependent DNA polymerases of both prokaryotic and eukaryotic origin by Bernad et al. (Bernad, A., Blanco, L., Lazaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228).

元の言語英語
ページ(範囲)14655-14660
ページ数6
ジャーナルJournal of Biological Chemistry
269
発行部数20
出版物ステータス出版済み - 1 1 1994

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DNA Polymerase II
Exonucleases
Amino Acid Motifs
Escherichia coli
Amino Acid Sequence
Amino Acids
DNA-Directed DNA Polymerase
Catalytic Domain
Nucleotides
Mutagenesis
Conserved Sequence
Sequence Alignment
DNA
Site-Directed Mutagenesis
DNA Replication
Elongation
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

Amino acid sequence motifs essential to 3' → 5' exonuclease activity of Escherichia coli DNA polymerase II. / Ishino, Y.; Iwasaki, H.; Kato, I.; Shinagawa, H.

:: Journal of Biological Chemistry, 巻 269, 番号 20, 01.01.1994, p. 14655-14660.

研究成果: ジャーナルへの寄稿記事

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AB - Many DNA polymerases have conserved sequences required for 3' → 5' exonuclease activity, which contributes to the accuracy of DNA replication by removing misincorporated nucleotides prior to chain elongation. Using amino acid sequence alignments, we predicted the putative active site of the 3' → 5' exonuclease of Escherichia coli DNA polymerase II. Site-directed mutagenesis at D155A, E157A, D155A/E157A, D228A, Y330F, and D334A, which are in the predicted exonuclease active regions, specifically inactivated 3' → 5' exonucleolytic activity but not DNA-polymerizing activity of E. coli DNA polymerase II. Furthermore, all of the mutants were diminished in the in vitro proofreading ability, as judged by their increased insertion and extension of wrong nucleotides. These findings indicate that the 3' → 5' exonuclease region of the E. coli DNA polymerase II is in the amino-terminal part of the protein, as it is in other DNA polymerases, and are consistent with the proposal of an evolutionary conserved 3' → 5' exonuclease active site in most DNA-dependent DNA polymerases of both prokaryotic and eukaryotic origin by Bernad et al. (Bernad, A., Blanco, L., Lazaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228).

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