Enzymic Unwinding of DNA

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Abstract
In connection with DNA, ATP dephosphorylation probably can be used to provide energy for mechanical effects. A novel DNA-dependent ATPase [EC 3.6.1-] purified to 90% homogeneity from E. coli was studied. The enzyme has a MW .apprx. 180,000 and in high salt it is a monomeric and probably highly anisometric molecule. In salt-free buffer where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km = 0.27 mM) and dephosphorylated at the .gamma.-position at a maximal rate near 104 molecules/enzyme monomer per min at 35.degree. C. A divalent cation requirement is best satisfied by Mg2+ or Ca2+, and the DNA requirement is best satisfied by the single-stranded, circular DNA of phage .PHI.X174 (Km = 62 nM nucleotide) and phage fd, indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein, .PHI.X DNA is not accepted, but once in contact with the DNA the enzyme is weakly inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the DNA recognition. The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the E. coli K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB-product-associated ATPase or the ATPase activity found in DNA polymerase III holoenzyme under appropriate conditions. It is not identical with a DNA-dependent ATPase of MW 69,000 from E. coli which was recently purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, failed.