Proofreading Exonuclease Research Articles

Interaction of the carcinogen N-acetoxy- N-2-acetylaminofluorene with polyadenylic acid: dependence of reactivity on conformation

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 polκ. Human polκ 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 polκ possesses optimal activity at 37 °C over the pH range 6.5–7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mm. Either Mg2+ or Mn2+ can satisfy a metal cofactor requirement for polκ activity, with Mg2+ being preferred. Human polκ is unable to bypass a cisplatin adduct in the template. However, polκ 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 polκ 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.