HeLa cell DNA polymerase .gamma.: further purification and properties of the enzyme

Abstract

DNA polymerase gamma has been purified over 60 000-fold from HeLa cells which contain no detectable type C viral particles. This purified enzyme shows a specific activity of 25 000 units/mg of protein which is comparable to the known specific activity of homogeneous preparations of human alpha and beta polymerases. The isolated enzyme shows apparent molecular weights ranging from 160 000 to 330 000 according to the method of analysis. The enzyme exhibits optimal activity for copying poly(A) in the presence of 50 mM KPO4 and 130 mM KCl and, under these conditions, copies poly(A) 20 times more rapidly than activated DNA. These assay conditions permit a clear distinction between the gamma-polymerase and DNA polymerase beta which is markedly inhibited by phosphate at this concentration. A comparison of the copying of activated DNA, poly(dA) and poly(A) by DNA polymerases alpha, beta, and gamma under optimal assay conditions for each enzyme is presented. Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.

Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma

“…The DNA polymerase gamma activity was eluted at a phosphate buffer concentration of 0.31 to 0.35 M and was totally separated from any residual DNA polymerase alpha activity carried over from the Q-Sepharose column, which was eluted between 0.12 and 0.17 M phosphate buffer. The concentration of phosphate buffer required to elute the DNA polymerase activity from this column was almost identical to that reported previously (14). This observation, together with the preparations' ability to use poly(rA) -poly(dT)1218 as template and primer in the presence of 50 mM potassium phosphate buffer (pH 7.5) and Km values for dNTPs (see below), was used as evidence that the DNA polymerase activity was that of a gamma polymerase.…”

“…DNA polymerase gamma was partially purified from HeLa Ohio cells by chromatography on Q-Sepharose Fast Flow; this was followed by chromatography on double-stranded DNA cellulose; the conditions used were based on those described previously (14). The major differences in the procedure from that described previously (14) were that all buffers contained 0.5 mM ?henylmethylsulfonyl fluoride and 10 p,g of pepstatin A ml-, and fractions from the doublestranded DNA cellulose column were collected in tubes containing 0.1 ml of a 15-mg/ml solution of bovine serum albumin, such that its final concentration was approximately 0.6 mg/ml.…”

“…The major differences in the procedure from that described previously (14) were that all buffers contained 0.5 mM ?henylmethylsulfonyl fluoride and 10 p,g of pepstatin A ml-, and fractions from the doublestranded DNA cellulose column were collected in tubes containing 0.1 ml of a 15-mg/ml solution of bovine serum albumin, such that its final concentration was approximately 0.6 mg/ml. If bovine serum albumin was not used, much of the polymerase activity was lost.…”

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

“…DNA polymerase Y was isolated according to Knopf et al (15) and purified by DEAE cellulose, phosphocellulose, hydroxylapatite and DNA cellulose chromatography. The enzyme was essentially free from contaminating DNA polymerase a or B as indicated by its sensitivity to N-ethylmale4mide and 99% inhibition by ddTTP under conditions which do not inhibit DNA polymerase a or B (11).…”

DNA polymerase alpha mutants from a Drosophila melanogaster cell line.

“…Another resistant mutant appears to overproduce DNA polymerase a about 10-fold. A possible overproducing mutant has been described (14) (20). One unit of activity is the amount that catalyzes the incorporation of 1 nmol of dNTPs into acid-insoluble material in 60 min at 300C.…”