Abstract
Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.
Highlights
Its DNA from the host cell (F’) to an F- recipient cell which is in direcpthysical contact with the host(for a recent review, see Ref. 20)
Helicase and ssDNA-dependent ATPase Reactions-The unwinding reaction catalyzed by helicase I was originally characterized using either acoupled assay that measured the fraction of a radioactively labeled DNA substrate rendered susceptible to S1 nuclease, or by velocity sedimentation of the DNA substrate [18,22,23].We have extended this characterization using an assay that directly measures the ability of helicase I to unwind a partialduplex DNA molecule[24].This assay has been used to characterize several other DNA helicases [5, 24,32,33]
Since helicase I does not appear to turn overfrom one circular DNA substrate molecule to another, we investigated ing 2.5 kb of duplex DNA.3These resultssuggest that helicase I catalyzes a processive unwinding reaction
Summary
Purification of Helicase I-Helicase I was purified as described under “Experimental Procedures”; Table I summarizes the purification. The initial cell extract contained multiple DNA-dependent as well as DNA-independent ATPase activities making it impossible to estimate the total helicase I activity in crude extracts For this reason, no estimate of overall yield is made. DNA Substrate Requirements for ATP Hydrolysis-The helicases characterized to dateare all ssDNA-dependent ATPases [1].Table I11 summarizes the results of experiments performed using several different DNA moleculesas effectors of the helicase I-catalyzed ssDNA-dependent ATPase reaction. Neither poly(dT) nor linear M13mp ssDNA could serve as effectors of the ATP hydrolysis reaction Since both are ssDNA molecules we expected that they would substitute for circular ssDNA. These results suggest that DNA termini may inhibit the ssDNA-dependent ATPase reaction catalyzed by helicase I. ATPase activity wasmeasured in the standard ATP hydrolysis assay, using the indicated DNA effector, as described under “Experimental Procedures”using 10.5 ng of helicase I
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