Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight

Abstract

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an 'activated'-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5'-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 X 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

Aspects of Thymidine Metabolism and Function in Cultured Mammalian Cells Infected with Herpes Simplex Virus Type 1

“…6) occurs at about 5 /~M-dTTP, thus supporting the above suggestion that the polymerase has highest affinity for this substrate when the cellular concentration is low, before the virus-induced thymidine kinase activity can increase the pool size. Non-hyperbolic DNA polymerase kinetics have previously been reported for the unicellular green flagellate Euglena graeilis (McLennan & Keir, 1975). The stimulation of polymerases A and B (authors' nomenclature), which occurred at high concentrations of deoxyribonucleoside triphosphates, was attributed to substrate activation, and allosteric control of the enzymes was considered as a possible reason for this.…”

Partial purification and characterization of DNA polymerases from the cauliflower inflorescence.

“…She postulated that DNA polymerase-jl may have evolved when animal cells developed a multicellular form. McLennan et al (1975) reported that two DNA polymerases, pol A and B, from the unicellular alga, Euglena gracilis have similar characteristics and high molecular weights. Ross and Harris (1978) also reported that Chlamydomonas reinhardii has high molecular weight DNA polymerases, A and B and that a 3 S species was found in aged preparations of both enzymes.…”

DNA Polymerases a and y during Pre‐emergence and Early Larval Development of Artemia

“…Assay volumes were 150 p1 and incubations were for 30 min at 37 'C. Acid-insoluble radioactivity was determined as previously described [12]. 1 unit of enzyme activity is that amount which catalyses the incorporation of 1 nmol deoxynucleoside monophosphates into acid-insoluble material in 30 min at 37°C.…”

“…The unbound activity responding to assay A in Fig. 1 [12][13][14][15][16][17] embryo extract on Cihucron blue 3-GA Mutrex gel. Chinese cysts were incubated for 12 h and an extract prepared as described in Materials and Methods.…”