Purification and Characterization of Two DNA Helicases from Calf

Two DNA helicases from calf thymus nuclei have been purified and characterized. The two proteins, designated as nuclear DNA helicase I and 11, were copurified on Bio-Rex 70, DEAE-Sepharose, phosphocellulose and subsequently separated from each other on a heparin-Sepharose column. Final purification of DNA helicase I was achieved on single-stranded DNAcellulose and that of DNA helicase I1 on ATP-agarose. On denaturing polyacrylamide gels, nuclear DNA helicase I displayed two polypeptide bands of 170 and 200 kDa; nuclear DNA helicase I1 also consisted of two bands, in this case of 100 and 130 kDa. Both enzymes catalyzed the unwinding of a DNA primer from a single-stranded DNA template but had different nucleotide requirements for their energy supply. While nuclear DNA helicase I preferred to hydrolyze ATP or dATP to support its unwinding activity, nuclear DNA helicase I1 could utilize all four rNTPs or dNTPs, though ATP or dATP were still preferred to other nucleotides. ADP, AMP, or adenosine B’-O-(thiotriphosphate) could not be used by either enzyme in the unwinding reactions. A divalent cation was essential for activity of both enzymes. Nuclear DNA helicase I required 3-6 mM M e or 1 mM Mn2+ for optimal unwinding. In contrast, nuclear DNA helicase I1 displayed high activity even at very low concentrations of M 8 + . Nuclear DNA helicase I was stimulated by NaCl, KCl, or potassium acetate up to concentrations of 160 mM; in contrast, nuclear DNA helicase I1 was strongly inhibited at salt concentrations over 75 mM. Both DNA helicases had an associated ATPase activity. However, while the ATPase activity of nuclear DNA helicase I was stimulated only in presence of singlestranded DNA, the ATPase activity of the nuclear DNA helicase I1 was stimulated by single-stranded DNA and, even more efficiently, by RNA. Finally, the translocation of both DNA helicases had a polarity from 3’ to 5’ with respect to the single-stranded DNA template to which the enzymes were bound. A comparison of these DNA helicases with the other reported mammalian DNA helicases will be given. The significance of the association of the two DNA helicases during the process of the purification will be discussed.