Purification and characterization of pol kappa, a DNA polymerase encoded by the human DINB1 gene.


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.



Exonuclease Activity, Source / Purification, Nucleotide Analogs / Template Lesions, Nucleotide Incorporation


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Polymerase Reference Property Result Context
Human Pol kappa Gerlach VL2001 3-5' Exonuclease (proofreading) No
Human Pol kappa Gerlach VL2001 Cloned or native Insect cells
Human Pol kappa Gerlach VL2001 Template lesions Bypasses Reaction: Nucleotide incorporation
Human Pol kappa Gerlach VL2001 Tagged Yes
Human Pol kappa Gerlach VL2001 Tag Name GST
Human Pol kappa Gerlach VL2001 Full length or truncated Full length
Human Pol kappa Gerlach VL2001 Processivity 1bp

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