The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 M and in a temperature range between 4 and 37°C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5-adenylyl-␤,␥-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and singlestranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.Genetic information is stored into the double-stranded DNA molecule that is stabilized through specific hydrogen-bonded base pairs. However, replication and repair as well as recombination of the DNA molecule require a single-stranded DNA to be available at least transiently. In cells, unwinding and separation of the complementary strands of duplex DNAs (dsDNA) 1 into single-stranded DNAs (ssDNA) are catalyzed by a class of enzymes known as helicases. DNA helicases destabilize and unwind the duplex DNA through a series of energetic states, driven by the binding and hydrolysis of NTP, usually ATP, and subsequent release of NDP and inorganic phosphate. Thus, DNA helicases convert the chemical energy into mechanical energy for DNA unwinding and translocation along the nucleic acid lattice (for reviews, see Refs. 1-3).During unwinding of dsDNA, and to translocate processively without dissociation from DNA, the helicase must use at least two DNA-binding sites to keep contact with the DNA lattice; one binds to ssDNA for translocating, whereas the other binds to dsDNA for DNA unwinding. These two DNA binding sites may be located at different domains within a single polypeptide of a monomer or be held by two different polypeptides within a dimer or an oligomer for providing multiple DNA-binding sites. Corresponding to the first possibility, the "inchworm" model was proposed, in which the helicase is assumed to possess two nonidentical DNA binding sites (4); the "leading" site binds both ssDNA and dsDNA and interacts with the duplex to be unwound during s...

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