YEAST DNA HELICASE-A - CLONING, EXPRESSION, PURIFICATION, AND ENZYMATIC CHARACTERIZATION

We have cloned and expressed the yeast DNA helicase A in Escherichia c oli at a high level (similar to 30 mg/L of culture) in soluble form. W e describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme. In denaturing gel electro phoresis the enzyme behaved as a similar to 90 kDa protein, The native structure, determined by gel-filtration studies, appeared to be hexam eric and its quaternary structure was salt (NaCl) dependent. In low-sa lt buffers (containing 50 mM NaCl), the enzyme eluted in a single acti vity peak at an elution volume that appeared to correlate with a possi ble hexameric structure, In higher salt buffer (containing greater tha n 150 mM NaCl), the enzyme eluted as smaller assemblies (monomer/dimer ). The recombinant helicase A was able to hydrolyze ATP or dATP with e qual efficiency, The ATPase activity of the enzyme was absolutely DNA- dependent. The nucleotidase activities were comparable to those of the native enzyme. Kinetic analysis of the ATPase activity demonstrated t hat the K-m of the enzyme was similar to 90 mu M and the rate of ATP h ydrolysis was similar to 20 ATP s(-1) molecule(-1). DNA sequences cont aining pyrimidine stretches were more effective activators than those containing purine stretches. However, poly(dC) appeared to be the most effective activator of the ATPase activity. The ATPase activity was i nhibited by salt (NaCl) above 50 mM with a half-maximal inhibition at similar to 110 mM. It is likely that the active state of helicase A is hexameric. The helicase activity of the recombinant enzyme was stimul ated significantly by the yeast replication protein A (RPA) and to a l ower extent by the single-stranded DNA binding protein of E. coli (SSB ). The DNA helicase migrated on a DNA template in a 5' --> 3' directio n, Helicase A appeared to share a number of enzymatic characteristics including directionality, stimulation by RPA/SSB, and quaternary struc ture (monomer-hexamer) dynamics that are common to known replicative h elicases such as DnaB helicase and the SV40 T-antigen.