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J Biol Chem, Vol. 274, Issue 45, 31896-31902, November 5, 1999

The Kinetic Mechanism of EcoRI Endonuclease

From the Department of Biochemistry and ^¶ Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

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.

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