A low molecular weight DNA polymerase from ovaries of the frog Xenopus laevis. DNA polymerase-beta (ovarian).

A low molecular weight DNA polymerase (DNA nucleotidyltransferase; EC 2.7.7.7) has been purified 265,000-fold from ovaries of the frogXertopus Zuevis. On polyacrylamide gels run under denaturing conditions the purified preparation exhibited one major band of approximately 45,000 molecular weight. The most purified fraction incorporated 380 pmol of dTMP/h/mg of protein at 26”. No endodeoxyribonuclease, exodeoxyribonuclease, ribonuclease, or ribonuclease H activity was detected in the most purified fraction. The purified activity exhibited a Stokes’ radius of 29.5 + 1 A, as determined by gel filtration on Sephadex G-100, and a sedimentation coefficient of 3.5 S, determined by zone sedimentation in sucrose gradients. From these parameters and assuming a partial specific volume of 0.74 cmYg a native molecular weight of 45,500 was calculated using the SiegelMonty relationship. The purified activity exhibited an optimum at pH 8.7 to 9.1 and was stimulated by 0.1 M NaCl, KCl, or CsCl. With poly(A) oligo(dT) templates, the purified activity was absolutely dependent upon Mn2+ ions and was inhibited by Mg2+ ions. In contrast, activity with poly(dA).oligo(dT) and activated DNA templates utilized either Mn’+ or MgZ+ as cofactor. The purified DNA polymerase was inactivated by preincubation with 30 FM p-chloromercuribenzoate, but not by N-ethylmaleimide at concentrations up to 10 mn. Most of the properties of the low molecular weight DNA polymerase purified from ovaries of X. laeuis are consistent with the enzyme being a polymerase of the /I type.