The purification and characterization of the Escherichia coli helicase II protein and a study of the protein's enzymatic activities

Helicase II protein is essential in both methyl-directed mismatch and uvrABC excision DNA repair and may have other functions in DNA replication and recombination. In order to facilitate the isolation of the helicase II protein, I have subcloned the uvrD gene into a temperature-inducible expression system (Shimatake, H. and Rosenberg, M. (1981) Nature 292, 128-132.). The uvrD gene product is overexpressed to {dollar}sim{dollar}1-2% of total cellular protein by this system. A procedure for the large-scale purification of the Escherichia coli encoded helicase II protein (uvrD gene product) is reported with yields of {dollar}sim{dollar}60 mg of helicase II protein ({dollar}>{dollar}99% pure) from 250 grams of E. coli cells that overexpress the uvrD gene product. The solubility of helicase II protein was measured as a function of (glycerol), (NaCl) and pH as well as the presence of MgCl{dollar}sb2,{dollar} ATP or ADP at 25{dollar}spcirc{dollar}C and 37{dollar}spcirc{dollar}C. The aggregation state of helicase II protein was examined under the solution conditions used to investigate the protein's DNA unwinding activity, and the protein appears to be monomeric in these conditions. The helicase properties of this enzyme demonstrate an apparent 3{dollar}spprime{dollar} to 5{dollar}spprime{dollar} polarity, yet this enzyme can efficiently unwind duplex DNA from blunt-ends and at nicks in some conditions, in vitro. A spectroscopic assay that continuously measures the hydrolysis of ATP is also described and used to monitor DNA unwinding on different DNA substrates in the presence of helicase II protein. An analysis of this study demonstrated that DNA unwinding initiates as efficiently at the ends of DNA substrates containing 4 bases protruding from the 5{dollar}spprime{dollar} end of duplex DNA as substrates containing blunt-ends, yet the efficiency of initiating unwinding at the ends of substrates containing 4 bases protruding from the 3{dollar}spprime{dollar} end of duplex DNA or at nicked sites on nicked circular DNA was dramatically lower. Models for the initiation of DNA unwinding by helicase II protein on these DNA substrates as well as from a nick in a number of DNA repair processes are proposed.