Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction

Abstract

The heterogeneity of calf thymus DNA polymerase-.alpha. was further investigated. An enzyme (enzyme D) which exhibits higher activity on poly(dA) .cntdot. (dT)10 (A:T = 20:1) compared with that on activated DNA, was further purified and its properties compared with 2 other activities of the DNA polymerase-.alpha. fraction (enzymes A1 and C) which do not show a preference for poly(dA) .cntdot. (dT)10 over activated DNA. As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-.alpha. in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a MW of about 140,000, does not use a poly(A) .cntdot. (dT)10 template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behavior on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-.beta.. The extreme sensitivity of enzyme D to inhibition by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. Enzymes C and D are readily inactivated by heating at 45.degree. C unlike enzyme A1. The possible interrelationships of the multiple activities of calf thymus DNA polymerase-.alpha. are discussed.