FIG. 4.

SPP1 Chu digests linear dsDNA. PstI-cut pUC19 was digested under standard reaction conditions, fluorescently labeled, and quantified by excitation at 484 nm and scanning of the emission between 510 and 545 nm in a spectrofluorometer (QuantumMaster; see Materials and Methods). (A) Raw data obtained from the spectrofluorometer. Because the dye fluoresces with a higher intensity when bound to dsDNA than ssDNA or nucleotides, a decrease in fluorescence reflects digestion of DNA. Titrations were performed with various concentrations of refolded Chu against 2.3 nM DNA ends. Reaction time was 30 min. The dsDNA curve represents the fluorescence output from a reaction in which a quantity of EDTA sufficient to quench the reaction was added prior to the addition of enzyme. Curves 1 through 8 contained 20, 40, 60, 80, 120, 240, 160, and 200 nM Chu, respectively. The ssDNA curve represents data from a reaction mixture that contained 0.5 equivalent of denatured input DNA to imitate limit digest conditions. DNA products are illustrated to the right of the chart, with Chu represented as a circle with a slice missing. (B and C) Time course of DNA digestion by native Chu under saturation conditions initiated with enzyme and Mg²⁺, respectively. Samples were withdrawn and quenched with EDTA at various times. DNA and Chu concentrations were 2.3 nM DNA ends and at least 2.3 nM Chu (as determined empirically by titration). (D and E) Titrations of native and refolded Chu, respectively, against 2.3 nM DNA ends. Reaction time was 10 min.