Sequence-specific responses of restriction endonucleases to bromodeoxyuridine substitution in mammalian DNA.

AUTOR(ES)

Petruska, J

RESUMO

Substitution of BrdU for dT in mammalian DNA alters the rates of DNA cleavage by restriction endonucleases in a manner that can be related to the specificity of cleavage. A formula is proposed that describes inhibitory and stimulatory contributions arising from the substitution of a Br atom for the CH3 group on T. The larger Br atom is postulated to sterically hinder the nuclease from binding to adjacent groups in the DNA cleavage site, while allowing a tighter binding to itself. The inhibition caused by steric hindrance is predicted to vary inversely with distance from the point of cleavage, whereas the stimulation caused by tighter binding is predicted to be independent of distance. The resultant formula gives a good fit to the data obtained for thirteen different restriction nucleases of known specificity. The parameters in the formula appear to be simple functions of ionic strength. This formula can be used to predict the effect of BrdU substitution on any endonuclease whose specificity of cleavage is known.

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