DnaB helicase stimulates primer synthesis activity on short oligonucleotide templates

DnaB helicase stimulated the second-order RNA primer synthesis activity of primase by over 5000-fold on DNA templates that were 23 nucleotides long. T his template length is the same as the DnaB helicase thermodynamic binding site size [Jezewska, M. J., and Bujalowski, W. (1996) Biochemistry 35, 2117 -2128]. This phenomenal stimulation was achieved by increasing the template affinity of primase by over 300-fold and increasing the catalytic rate by over 15-fold. It was necessary to determine the optimal amount of DnaB heli case to achieve this stimulation because helicase stimulation was cooperati ve at low concentration and inhibitory at high helicase concentration The c ooperative stimulation at low concentration indicated the presence of a tim e-dependent assembly step that preceded the active state. Besides stimulati ng primase activity, DnaB helicase also prevented primase from synthesizing RNA primers that were longer than the template sequence. In the absence of DnaB helicase, the majority of primers synthesized by primase were longer than the template and were named "overlong primers" [Swart, J. R., and Grie p, M. A. (1995) Biochemistry 34, 16097-16106]. In contrast, the helicase-st imulated RNA primers were from 10 to 14 nucleotides in length with the 12-m er representing the majority of the total RNA primers produced. It was show n that DnaB helicase stabilized the open or single-stranded conformation of the template, which favored the synthesis of the template-length-dependent primers. In contrast, when primase acted alone, it stabilized the 3'-end h airpin conformation of the template so that the template's 3'-hydroxyl serv ed as a "DNA primer" from which primase elongated to create the overlong pr imers.