Expression of human DNA polymerase beta in Escherichia coli and characterization of the recombinant enzyme.

Abstract:

The coding region of a human beta-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with beta-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as beta-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural beta-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant beta-polymerase using defined template-primer systems. The results indicate that this beta-polymerase is essentially identical with natural beta-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol beta Abbotts J1988 Molecular Weight 3.9E+04 Dalton Technique: SDS-PAGE
Human Pol beta Abbotts J1988 3-5' Exonuclease (proofreading) No
Human Pol beta Abbotts J1988 Cloned or native Cloned in E. coli
Human Pol beta Abbotts J1988 5-3' Exonuclease No
Human Pol beta Abbotts J1988 Full length or truncated Full length
Human Pol beta Abbotts J1988 Processivity 8bp
Human Pol beta Abbotts J1988 Nick Extension No
Human Pol beta Abbotts J1988 Gap Filling No

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