Inhibition of restriction endonuclease activity by DNA binding fluorochromes.
Activity of type II restriction endonuclease is affected by many common factors including buffer composition and sequences flanking the recognition site (Brabec et al., Eur.J. Biochem. 216, 183, 1993). The successful development of Optical Mapping (Schwartz et al., Science, 262, 110, 1993; Meng et al., Nature Genet. 9, 432, 1995; Wang and Schwartz, PNAS, 1995 Cai et al., PNAS, 92, 5164, 1995) relied on optimization of light microscope-based imaging of fluorescently labeled DNA molecules during restriction endonuclease digestion. Little was known about the effects of commonly used DNA-fluorochromes on restriction endonuclease activity. Thus, we developed an enzyme activity assay using lambda bacteriophage DNA or adenovirus-2 DNA to evaluate the effects of five DNA binding fluorochromes (4'-6-daimidine-2-phenylindole (DAPI), ethidium bromide (EtdBr), ethidium bromide homodimer (EthD-1), bis-benzimide (H33258) and benzothiazolium-4-quinolinium dimer (TOTO-1)) on the enzymatic activities of eleven type II restriction endonucleases (Asc I, Csp I, Dra I, EcoR I, Hha I, Hind III, Not I, Rsr II, Sfi I, SgrA I and Sma I). We found that the minor groove binding fluorochrome, DAPI, did not measurably inhibit activity of this group, with the exception of Dra I. Similarly, another minor groove binding fluorochrome H33258 inhibited Dra I and Not I (slightly). The three intercalating fluorochromes EtdBr, EthD-1 and TOTO-1, however, variably inhibited the other enzymes. Since Beta-mercaptoethanol (Beta-ME) is used to discourage photodamage of stained DNA molecules, we also assessed its effect on restriction endonuclease activity. Interestingly, Dra I, Hind III, Sfi I and Sma I retained full activities at high concentration of Beta-ME (5%), but Asc I, Csp I, Not I, Rsr II and SgrA I showed varying sensitivities to the Beta-ME. Isoschizomers Csp I and Rsr II behaved differently to both fluorochromes and Beta-ME. The results presented here should provide a basis for further development of new Optical Mapping-based techniques requiring fluorescence labeling of other actively imaged enzymatic reactions.
Meng
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W. M. Keck Laboratory for Biomolecular Imaging, Department of Chemistry, New York University, New York 10003, USA.
Cai
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Schwartz
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D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008623:Mercaptoethanol / Q000737:chemistry / Q000494:pharmacology; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D015183:Restriction Mapping / Q000379:methods*
D005456:Fluorescent Dyes; D008623:Mercaptoethanol; D004247:DNA; D004720:Endonucleases