Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II).
We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
G T
GT
Runyon
I
I
Wong
T M
TM
Lohman
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011108:Polymers / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012995:Solubility
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011108:Polymers; C506744:rep protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases