XENOPUS-LAEVIS OVARIAN DNA HELICASE .1. A 3' TO 5' HELICASE THAT UNWINDS SHORT DUPLEXES

A novel DNA helicase isolated from Xenopus laevis ovaries [Poll, E. H. A.. & Benbow, R.M. (1988) Biochemistry 27, 8701-8706] was characteriz ed biochemically. The directionality of DNA unwinding was determined t o be 3' to 5'. A short 3' ssDNA tail adjacent to duplex DNA was requir ed for DNA unwinding; the minimum length of this tail was between four and nine bases. Only short duplex DNA regions were unwound: duplex DN A of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not. Longer duplex regions were unwound in the presence of Escheri chia coli single-strand DNA binding protein if, in addition, the duple x region was flanked by an unpaired 3' or 5' tail and the substrate re sembled a branched replicative intermediate. X. laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA. DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 1 25 mM for NaxPO4 or KxPO4. The ATP analog ATPgammaS inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPC P and AMPPNP moderately inhibited DNA unwinding activity and had littl e effect on the copurifying DNA-dependent ATPase activity. CTP was a r elatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dC TP, dGTP, or TTP showed moderate or no inhibition. The copurifying DNA -dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, d GTP, or TTP.