Abstract
Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spaced EcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a single EcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease.DNA interactions, reflecting facilitated diffusion processes, that occur prior to EcoRI sequence recognition and subsequent to DNA cleavage.
Highlights
Clease locates an EcoRI sequence and departs from a cleaved site (19 –22)
This work demonstrates that product release is ratelimiting for turnover at ionic strengths of 0.06 – 0.23 M and show that, variation of ionic strength has dramatic effects on Km and kcat, the kinetics of events that occur at the recognition sequence are essentially insensitive to variation of this parameter
We show that the protein remains associated with the polynucleotide product after cleavage, undergoing facilitated transfer between DNA sites so rapidly that the kcat for processive cleavage of a two-site substrate is the same as that for cleavage of an otherwise identical DNA containing a single EcoRI site
Summary
Clease locates an EcoRI sequence and departs from a cleaved site (19 –22). the effects of reaction parameters that are expected to affect the efficiency of facilitated diffusion have not been examined with respect to catalytic behavior of the enzyme. We show that the protein remains associated with the polynucleotide product after cleavage, undergoing facilitated transfer between DNA sites so rapidly that the kcat for processive cleavage of a two-site substrate is the same as that for cleavage of an otherwise identical DNA containing a single EcoRI site. These observations indicate that the steadystate behavior of the enzyme on natural substrates is dominated by nonspecific interactions, reflecting the significance of facilitated diffusion processes in the reaction mechanism. Chemical quench experiments have indicated that product release is rate-limiting for turnover on ColE1 and pBR322 plasmid DNAs under one set of experimental conditions [5, 15], and facilitated diffusion has been implicated in the paths by which the endonu-
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