We have cloned and expressed the @entity375 DNA helicase A in @entity526 at a high level (approximately 30 mg/L of culture) in soluble form. We describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme. In denaturing gel electrophoresis the enzyme behaved as a approximately 90 kDa protein. The native structure, determined by gel-filtration studies, appeared to be hexameric and its quaternary structure was salt ( @entity1219 ) dependent. In low-salt buffers (containing 50 mM @entity1219 ), the enzyme eluted in a single activity peak at an elution volume that appeared to correlate with a possible hexameric structure. In higher salt buffer (containing greater than 150 mM @entity1219 ), the enzyme eluted as smaller assemblies (monomer/dimer). The recombinant helicase A was able to hydrolyze @entity855 or @entity17699 with equal 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 that the Km of the enzyme was approximately 90 microM and the rate of @entity855 hydrolysis was approximately 20 @entity855 s-1 molecule-1. DNA sequences containing @entity7391 stretches were more effective activators than those containing @entity10043 stretches. However, @entity70093 appeared to be the most effective activator of the ATPase activity. The ATPase activity was inhibited by salt ( @entity1219 ) above 50 mM with a half-maximal inhibition at approximately 110 mM. It is likely that the active state of helicase A is hexameric. The helicase activity of the recombinant enzyme was stimulated significantly by the @entity375 replication protein A (RPA) and to a lower extent by the single-stranded DNA binding protein of @entity526 ( @entity14769 ). The DNA helicase migrated on a DNA template in a 5' --> 3' direction. Helicase A appeared to share a number of enzymatic characteristics including directionality, stimulation by RPA/ @entity14769 , and quaternary structure (monomer-hexamer) dynamics that are common to known replicative helicases such as DnaB helicase and the SV40 T-antigen.

XXXX DNA helicase A: cloning, expression, purification, and enzymatic characterization.

@entity10043 :: ('MESH:C030985', 'Chemical') :: ['purine'] @entity526 :: ('562', 'Species') :: ['Escherichia coli', 'E. coli'] @entity1219 :: ('CHEBI:26710', 'Chemical') :: ['NaCl'] @entity17699 :: ('CHEBI:16284', 'Chemical') :: ['dATP'] @entity14769 :: ('20466802', 'Gene') :: ['SSB'] @entity70093 :: ('MESH:C023826', 'Chemical') :: ['poly(dC)'] @entity375 :: ('4932', 'Species') :: ['yeast'] @entity7391 :: ('MESH:C030986', 'Chemical') :: ['pyrimidine'] @entity855 :: ('MESH:D000255', 'Chemical') :: ['ATP']
@entity375 :: (4932,Species) :: ['Yeast']
