Expression of the catalytic subunit of human DNA polymerase delta in mammalian cells using a vaccinia virus vector system
| Autor: | Geraldine L. Holt, Richard C. Condit, Peng Zhang, Yunquan Jiang, Marietta Y.W.T. Lee, Isabelle Frugulhetti |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Exonuclease
DNA polymerase Protein subunit Genetic Vectors Molecular Sequence Data Vaccinia virus DNA-Directed DNA Polymerase Biochemistry DNA polymerase delta Catalysis law.invention Cell Line law Chlorocebus aethiops Animals Humans Binding site Cloning Molecular Molecular Biology Polymerase DNA Polymerase III DNA Primers biology Base Sequence Cell Biology Molecular biology Recombinant Proteins Proliferating cell nuclear antigen Mutation biology.protein Recombinant DNA |
| Zdroj: | The Journal of biological chemistry. 270(14) |
| ISSN: | 0021-9258 |
| Popis: | The catalytic polypeptide of human DNA polymerase delta was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTM1 system. The recombinant human DNA polymerase delta was purified to homogeneity in two steps using an immunoaffinity column and a single-stranded DNA-cellulose column. Levels of expression were about 1% of soluble cytosolic protein. The recombinant catalytic subunit was fully active and exhibited enzymatic properties similar to that of the native two-subunit enzyme including the possession of an associated 3' to 5' exonuclease activity. Recombinant pol delta was stimulated by proliferating cell nuclear antigen (PCNA); however, the degree of stimulation was lower than that of the native human enzyme. Analysis of a double mutant of the catalytic subunit, H142R/F144S, showed that it had a greatly reduced sensitivity to PCNA, suggesting that the PCNA binding site of pol delta may be located in this region of the N terminus. |
| Databáze: | OpenAIRE |
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