Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D
| Autor: | Juhani E. Syväoja, Helmut Pospiech, Maarit Jokela, Juha Rouvinen, Anitta Eskelinen |
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| Rok vydání: | 2004 |
| Předmět: |
DNA Replication
Models Molecular Exonuclease Base Pair Mismatch DNA polymerase Archaeal Proteins Methanococcus DNA polymerase II Molecular Sequence Data DNA-Directed DNA Polymerase Substrate Specificity Enzyme Stability Genetics Magnesium Amino Acid Sequence Polymerase Klenow fragment Endodeoxyribonucleases DNA clamp biology DNA replication Articles Molecular biology Protein Structure Tertiary Protein Subunits Exodeoxyribonucleases Biochemistry Mutation biology.protein Proofreading Sequence Alignment |
| Zdroj: | Nucleic Acids Research. 32:2430-2440 |
| ISSN: | 1362-4962 |
| Popis: | The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase. |
| Databáze: | OpenAIRE |
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