A low molecular weight DNA polymerase beta in the sea urchin Strongylocentrotus purpurantus. Partial purification, properties, and changes in development.
A low molecular weight DNA polymerase which sediments at 3.3 S on sucrose gradients has been purified from total cell homogenates of rapidly dividing embryos of the sea urchin Strongylocentrotus purpuratus. In the presence of 2 mM N-ethylmaleimide, it is the major polymerase activity in whole cell homogenates when assayed with an oligo(dT)10.poly(dA)200 template; a template which it uses about 200 times more efficiently than activated DNA. The requirement for N-ethylmaleimide exists only in crude cell fractions where it acts to inhibit a template digesting nuclease activity. The polymerase is highly stable if maintained in the presence of 20% glycerol, is completely dependent on added template, and shows no end addition activity. The physical and enzymatic properties of this enzyme clearly distinguish it from the DNA polymerase previously described by Loeb (Loeb, L. A. (1969) J. Biol. Chem. 244, 1672-1681) which sediments as a high moeluclar weight (5.6 to 6.6 S) enzyme and prefers the activated DNA template. In addition, these two DNA polymerase enzymes show distinctive chromatographic properties using DEAE-cellulose and phosphocellulose columns as well as their sensitivity to N-ethylmaleimide. The properties of the low molecular weight polymerase indicate close similarity to the beta-polymerase isolated from mammalian cells. These low molecular weight enzymes are both sensitive to phosphate salt and able to utilize the artificial ribohomopolymer template oligo(dT)10.poly(rA)200. A quantitative analysis of the low molecular weight DNA polymerase during early embryonic development indicates that the activity of this enzyme increases at least 2-fold immediately following fertilization and again during early blastula stage (hatching). Such quantitative changes in a beta enzyme activity are in contrast to findings with the alpha-polymerase which remains constant during early development.
D000818:Animals; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D005260:Female; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D010063:Ovum / Q000378:metabolism; D012617:Sea Urchins / Q000201:enzymology*; D013045:Species Specificity; D013698:Templates, Genetic
D007527:Isoenzymes; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase