Characterization of a DNA Polymerase Activity in Cultured Human Melanoma Cells

While utilizing poly(2’-O-methylcytidylate) oligodeoxyguanylate ((Cm),*(dG)lz-18) to assay for DNA polymerase activity during fractionation of total cell extracts of cultured human, malignant cells, a new DNA polymerase activity called DNA polymerase Cm was identified in the human melanoma cell line A-375. T h i s activity, which was not associated with particles with the density of RNA tumor viruses, was purified from the cytoplasmic fraction some 13,000-fold by sequential chromatography on DEAE-cellulose, phosphocellulose, CM-Sephadex, and poly(Cm)-Sepharose. Purified DNA polymerase Cm copied (A), (dT)~z-ls, (&)no (d‘F)12-18, and activated calf thymus DNA 20-, 7-, and 3.5-fold more efficiently than (Cm),. (dG)12-18. DNA polymerase Cm had a sedimentation coefficient of 3.4 S in 0.2 M NaCl and a molecular weight of 50,000 as estimated by Sephadex 6-100 gel filtration in 0.2 M NaCl. With (Cm)n*(dG)lz-~s, (A)n*(dT)12-18, or (Wn*(d’%-,s as template-primer, DNA polymerase Cm had a divalent metal ion optimum of 0.8 to 1.2 m~ MnCl,, was sensitive to inhibition by salt (70 to 100% at 0.2 M NaCl) or Nethylmaleimide (60% at 0.01 m ~ ) , and had a pH optimum of 8.2 in ”is-HC1 buffer. We have thus f a r been unable to copy retrovirus genomic RNA or globin mRNA plus (dT)12-18 with the purified enzyme preparation. DNA polymerase Cm was not inhibited by antisera prepared against either primate retrovirus reverse transcriptase or human cell DNA polymerase a. In addition to A-375 cells, a cell line established from a human lung carcinoma (A-1188) was found to contain the enzyme. Human normal skin fibroblast and embryo fibroblast cells did not contain detectable amounts of DNA polymerase Cm.