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The ATP-dependent 180,000 molecular weight DNA unwinding enzyme of Escherichia coli has been subjected to further study. The results support the following conclusions. The enzyme, the largest soluble peptide in E. coli, is a fibrous protein. The typical large aggregates which the enzyme forms are ordered structures. The enzyme accepts double-stranded DNA as a substrate only when the duplex DNA is covalently linked to single-stranded DNA. It initiates on such DNA by binding to the single-stranded region. Denaturation, an effect induced by ATP, then follows in this pre-formed enzyme-DNA complex. Denaturation requires dephosphorylation of the ATP cofactor; the mere presence of a recognized nucleoside triphosphate does not induce the enzyme to unwind. Some 80 enzyme molecules are required to unwind the double-stranded section of an fd bacteriophage DNA molecule, the typical substrate. The number of enzyme molecules required is not reduced when a non-enzymatic, single-stranded DNA binding protein is added to facilitate chain separation. There is sufficient enzyme in an E. coli cell to satisfy the needs of unwinding several duplexes.
These results are interpreted in terms of the previously proposed scheme of processive enzymatic action: an ATP dephosphorylating enzyme proceeds “actively” along a bound DNA chain, thereby displacing a base-paired complementary chain. The unwinding agent is probably a multimeric form of the enzyme protein. The implications of this concept are discussed with respect to the mechanism that causes myosin to proceed along an actin filament.
