Purification and characterization of a DNA polymerase gamma from human trophoblast tissue

Graduation date: 1984DNA polymerase gamma has been purified over 67,500-fold from human trophoblast tissue. Purification was achieved through successive chromatographies on phosphocellulose, DEAE-cellulose, DNA-cellulose, and high pressure gel exclusion through Fractogel TSK HW55. This preparation yielded a specific activity of 81 units/mg of protein using the preferred template-primer, poly (A)-oligo (dT)₁₂₋₁₃. Analysis by SDS-polyacrylamide gel electrophoresis revealed a single polypeptide band with molecular weight 94,000 daltons, confirming the molecular weight estimate determined by gel exclusion. Sedimentation analysis in high ionic strength revealed an enzyme with molecular weight of 160-180,000 daltons, suggesting the enzyme exist as a dimer of the 94,000 dalton polypeptide. The enzyme exhibits optimal activity with poly (A)-oligo (dT)₁₂₋₁₈ in the presence of 10 mM KPO₄, 90 mM KC1 and 0.5 mM Mn²⁺. This demonstrates the first structural analysis of a DNA polymerase gamma from a human origin