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.
Gerlach
V L
VL
Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9072, USA.
Feaver
W J
WJ
Fischhaber
P L
PL
Friedberg
E C
EC
D000099:Acetoxyacetylaminofluorene / Q000378:metabolism / Q000494:pharmacology; D000477:Alkylating Agents / Q000378:metabolism / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D002945:Cisplatin / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013698:Templates, Genetic; D000099270:Y-Family DNA Polymerases
D000099:Acetoxyacetylaminofluorene; D000477:Alkylating Agents; D002945:Cisplatin; D018736:DNA Adducts; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D011993:Recombinant Fusion Proteins; C057270:cisplatin-DNA adduct; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases