NUCLEAR-DNA HELICASE-II UNWINDS BOTH DNA AND RNA

Nuclear DNA helicase II (NDH II) has been purified to near-homogeneity by exploiting its high affinity to poly[(rI).(rC)]-agarose. The purif ied enzyme was obtained as two catalytically active forms of 130- and 100-kDa molecular mass, respectively. After treatment with cyanogen br omide, the separated polypeptides displayed very similar digestion pat terns. Thus, the 100-kDa form most likely is a proteolytic product of the 130-kDa polypeptide. For DNA unwinding, NDH II could use any of th e four rNTPs or dNTPs with K(m) values between 20 and 100 muM. DNA unw inding was stimulated up to 20-fold by substrates that contained singl e-stranded 3'-tails. NDH II-catalyzed DNA unwinding was strongly inhib ited by RNA, but was little affected by DNA. The strongest RNA inhibit or, poly[(rI).(rC)], was also the strongest effector of the NTPase act ivity of NDH II. The binding constant for poly[(rI).(rC)] binding was about 2 x 10(7) M-1; the minimal binding site size was determined as 1 6 nucleotides. In agreement with its high affinity to RNA, NDHII unwou nd double-stranded RNA. RNA unwinding required the presence of a nucle oside triphosphate and a divalent cation (Mg2+). Thus, like the protot ypic replicative helicase large T antigen of simian virus 40, NDH II m ay function in both DNA and RNA unwinding.