restriction endonucleases stui  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: A simple and general approach to generate photoactivatable DNA processing enzymes

doi: 10.1093/nar/gkab1212

Figure Lengend Snippet: Selective activation of the restriction enzyme StuI. ( A ) Digestion of lambda DNA by the StuI restriction enzyme. Efficient digestion of lambda DNA was observed by commercial StuI, unmodified StuI, and irradiated StuI-PC_oligo (lanes 2, 3 and 5, respectively). Interestingly, in the non-photoactivated StuI-PC_oligo sample (lane 4), the lambda DNA stays mostly uncut (see lane 1 for only lambda DNA reference), thus probing effective activation of the StuI-PC_oligo by light. C in lane 2 denotes control digestion and StuI is represented schematically as a scissor. ( B ) Digestion of a quencher-fluorophore dsDNA probe bearing a StuI restriction site. The irradiated StuI-PC_oligo samples (blue symbols) showed a fast increase of the fluorescence signal, consistent with the efficient digestion of the probe. This behaviour was largely suppressed in non-illuminated samples (red symbols), where just a mild increase in the fluorescence is observed when compared with dsDNA probe alone (black symbols). The observed digestion was sequence-specific, as the digestion of a dsDNA probe methylated in the restriction site (green symbols, see ) by the activated StuI_oligo was abolished. A light pulse of 120 s with 365nm wavelength UV light was used for both types of experiments.

Article Snippet: The activity of the restriction endonuclease StuI was measured in both phage lambda DNA (New England Biolabs, USA) and a quencher-fluorophore dsDNA probe.

Techniques: Activation Assay, Lambda DNA Preparation, Irradiation, Fluorescence, Sequencing, Methylation