Abstract
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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- Berkner K. L., Folk W. R. The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases. J Biol Chem. 1979 Apr 10;254(7):2551–2560. [PubMed] [Google Scholar]
- Bick M. D., Davidson R. L. Total substitution of bromodeoxyuridine for thymidine in the DNA of a bromodeoxyuridine-dependent cell line. Proc Natl Acad Sci U S A. 1974 May;71(5):2082–2086. doi: 10.1073/pnas.71.5.2082. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blin N., Stafford D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976 Sep;3(9):2303–2308. doi: 10.1093/nar/3.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goeddel D. V., Yansura D. G., Winston C., Caruthers M. H. Studies on gene control regions. VII. Effect of 5-bromuracil-substituted lac operators on the lac operator-lac repressor interaction. J Mol Biol. 1978 Aug 25;123(4):661–687. doi: 10.1016/0022-2836(78)90211-5. [DOI] [PubMed] [Google Scholar]
- Lin S. Y., Riggs A. D. Lac operator analogues: bromodeoxyuridine substitution in the lac operator affects the rate of dissociation of the lac repressor. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2574–2576. doi: 10.1073/pnas.69.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marchionni M. A., Roufa R. J. Digestion of 5-bromodeoxyuridine-substituted lambda-DNA by restriction endonucleases. J Biol Chem. 1978 Dec 25;253(24):9075–9081. [PubMed] [Google Scholar]
- Petruska J., Horn D. Bromodeoxyuridine substitution in mammalian DNA can both stimulate and inhibit restriction cleavage. Biochem Biophys Res Commun. 1980 Oct 16;96(3):1317–1324. doi: 10.1016/0006-291x(80)90095-9. [DOI] [PubMed] [Google Scholar]
- Polisky B., Greene P., Garfin D. E., McCarthy B. J., Goodman H. M., Boyer H. W. Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3310–3314. doi: 10.1073/pnas.72.9.3310. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rutter W. J., Pictet R. L., Morris P. W. Toward molecular mechanisms of developmental processes. Annu Rev Biochem. 1973;42:601–646. doi: 10.1146/annurev.bi.42.070173.003125. [DOI] [PubMed] [Google Scholar]
- Smith H. O. Nucleotide sequence specificity of restriction endonucleases. Science. 1979 Aug 3;205(4405):455–462. doi: 10.1126/science.377492. [DOI] [PubMed] [Google Scholar]
- Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]