Purification and characterization of pol kappa, a DNA polymerase encoded by the human DINB1 gene
- PMID: 11024016
- DOI: 10.1074/jbc.M004413200
Purification and characterization of pol kappa, a DNA polymerase encoded by the human DINB1 gene
Abstract
The Escherichia coli dinB gene encodes DNA polymerase (pol) IV, a protein involved in increasing spontaneous mutations in vivo. The protein-coding region of DINB1, the human ortholog of DNA pol IV, was fused to glutathione S-transferase and expressed in insect cells. The purified fusion protein was shown to be a template-directed DNA polymerase that we propose to designate pol kappa. Human pol kappa lacks detectable 3' --> 5' proofreading exonuclease activity and is not stimulated by recombinant human proliferating cell nuclear antigen in vitro. Between pH 6.5 and 8.5, human pol kappa possesses optimal activity at 37 degrees C over the pH range 6.5-7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mm. Either Mg(2+) or Mn(2+) can satisfy a metal cofactor requirement for pol kappa activity, with Mg(2+) being preferred. Human pol kappa is unable to bypass a cisplatin adduct in the template. However, pol kappa shows limited bypass of an 2-acetylaminofluorene lesion and can incorporate dCTP or dTTP across from this lesion, suggesting that the bypass is potentially mutagenic. These results are consistent with a model in which pol kappa acts as a specialized DNA polymerase whose possible role is to facilitate the replication of templates containing abnormal bases, or possessing structurally aberrant replication forks that inhibit normal DNA synthesis.
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