The Occurrence of Multiple Activities in the High‐Molecular‐Weight DNA Polymerase Fraction of Mammalian Tissues

Abstract

The high-molecular-weight DNA polymerase fraction of calf thymus, rat liver or rat spleen can be resolved into further activities differing from one another in molecular weight and charge properties. The use of Sepharose 6B columns brings about partial resolution of these activities although DEAE-cellulose achieves a more effective separation. The high-molecular-weight fraction from calf thymus has so far yielded four species of activity, A, B, C and D sedimenting at 7.9 S, 5.2 S, 7.3 S and 6.0 S, respectively. In rat liver and spleen, 7.6-S (I), 7 . 2 3 (11) and 5-S enzymes have been detected although smaller and more variable amounts of 5-S enzyme occur in the rat tissues compared with calf thymus where the 5.2-5 species can represent 20-

The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from Physarum polycephalum

“…171 enzyme may undergo a conformational transition to a much more elliptical shape in discrete steps (Table 1). These findings are consistent with the growing acceptance of heterogeneity of DNA polymerase activity, isolated from cytoplasmic extracts (Holmes et al, 1974;McLennan & Keir, 1975 Hecht (1973); the evaluation ofgel-filtration characteristics ofPhysarum cytoplasmic DNA polymerase activity before and after treatment with (NH4)2SO4 confirm this finding. The purification procedure of cytoplasmic DNA polymerase activity indicates that the column chromatography, carried out by the three columns in series (method 1), is a powerful tool to obtain a rapid and acceptable yield of a protein preparation that contains a drastically decreased number of proteins p (Fig.…”

Differential N‐ethylmaleimide inhibition of two enzymes of the DNA α‐polymerase‐fraction from calf thymus

“…In some preparations a further enzyme, B (5. 2 S), is found and probably arises proteolytically from enzyme C [2,4]. A fourth species of enzyme, D, has also been detected by its response to the synthetic template poly(dA) .…”

“…Calf thymus DNA a-polymerase was prepared and resolved into its subfractions by DEAE-cellulose chromatography as previously described [2] . In the present studies enzymes Ai (referred to in the text as enzyme A) and C were taken and chromatographed a second time on DEAE-cellulose before use (specific activities were 1300 and 2300 units/mg protein, respectively).…”

“…Under the conditions of the assay (containing 1 mM dithiothreitol), incorpora-tion of [ 3H] dTMP was linear with time for at least 20 min. The standard DNA polymerase assay using activated DNA as template was performed as described [2,11] . For convenience in time courses, assay incubation time was 11 min.…”

DNA polymerases of eukaryotes

“…Heterogeneity of the enzyme activity has been observed in a variety of tissues and cells such as calf thymus [34,54,55], rat liver [34], mouse myeloma [56,57] BHK cells [58] and rabbit bone marrow [59]. This heterogeneity may be due to aggregation [26], proteolysis or possibly to association with other proteins of the replication complex and these probably account for some of the difficulties in purification.…”