title: Yeast DNA helicase A: cloning, expression, purification, and enzymatic characterization.
abstract: We have cloned and expressed the yeast DNA helicase A in Escherichia coli 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 (NaCl) dependent. In low-salt buffers (containing 50 mM NaCl), 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 NaCl), the enzyme eluted as smaller assemblies (monomer/dimer). The recombinant helicase A was able to hydrolyze ATP or dATP 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 ATP hydrolysis was approximately 20 ATP s-1 molecule-1. DNA sequences containing 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 inhibited by salt (NaCl) 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 yeast replication protein A (RPA) and to a lower extent by the single-stranded DNA binding protein of E. coli (SSB). 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/SSB, and quaternary structure (monomer-hexamer) dynamics that are common to known replicative helicases such as DnaB helicase and the SV40 T-antigen.
authors.affiliation: Department of Molecular Biology, Science Center, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
authors.forename: E E
authors.initials: EE
authors.lastname: Biswas
authors.forename: W M
authors.initials: WM
authors.lastname: Fricke
authors.forename: P H
authors.initials: PH
authors.lastname: Chen
authors.forename: S B
authors.initials: SB
authors.lastname: Biswas
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D053488:DnaB Helicases; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C506734:HCS1 protein, S cerevisiae; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases