Rates of chemical cleavage were determined by mixing UNG and either substrate 3U () or 4U ()

<p><b>Copyright information:</b></p><p>Taken from "A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping"</p><p></p><p>Nucleic Acids Research 2007;35(5):1478-1487.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865060.</p><p>© 2007 The Author(s).</p> The observed rates () are plotted against enzyme concentration. ( Data for the AT-rich single-stranded oligonucleotide 3U is shown with the best fit to Equation (), with values of = 37.5 ± 1.8 s and = 3.9 ± 0.5 μM. () The data for the GC-rich single-stranded oligonucleotide 4U did not reach saturation and exhibited a linear rather than hyperbolic relationship, hence is shown with the best fit to a linear equation

Fluorescence states were determined by titrating increasing concentrations of oligonucleotides 1U (panels and ) and 2U (panels and ), or fixed ratios of oligonucleotides and enzyme, and observing the change in fluorescence

<p><b>Copyright information:</b></p><p>Taken from "A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping"</p><p></p><p>Nucleic Acids Research 2007;35(5):1478-1487.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865060.</p><p>© 2007 The Author(s).</p> All data are shown with the best fit to a linear equation. Fluorescence states were assigned for free substrate (open circles, red line), the specific E·S complex (open triangles, cyan line), the free abasic product (open diamonds, green line), the E·P complex with both wild-type UNG (closed circles, maroon line) and the D88N/H210N mutant (closed triangles, dark green line). The control oligonucleotide (1N) with 2-AP not adjacent to the target uracil was also examined as free DNA (open squares, magenta line), and in an enzyme–DNA complex (open inverted triangles, black line)

Substrates 1HU (left column) and 2HU (right column) were mixed with increasing concentrations of D88N/H210N UNG using stopped-flow

<p><b>Copyright information:</b></p><p>Taken from "A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping"</p><p></p><p>Nucleic Acids Research 2007;35(5):1478-1487.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865060.</p><p>© 2007 The Author(s).</p> Anisotropy ( and ) and total HEX fluorescence ( and ) were simultaneously monitored, and the same solutions were then used to collect 2-AP fluorescence ( and ). The data are shown with the results of a global fit to Scheme 1. Individual curves for each of the enzyme concentrations used are shown: 8 μM (red); 3 μM (green); 2 μM (blue); 1 μM (cyan); 0.5 μM (magenta) and 0.2 μM (purple), all reactions were performed with 0.1 μM substrate and other conditions as described in the Materials and methods section

A complete reaction cycle of UNG was analysed by monitoring 2-AP fluorescence using stopped-flow to rapidly mixing equimolar amounts of wtUNG and substrates 1U () and 2U () at concentrations in excess of the (4 μM 1U and 20 μM 2U)

<p><b>Copyright information:</b></p><p>Taken from "A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping"</p><p></p><p>Nucleic Acids Research 2007;35(5):1478-1487.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865060.</p><p>© 2007 The Author(s).</p> The data are shown with the best fit to Scheme 1 using kinetic parameters determined from the global stopped-flow analysis (), the cleavage rate determined from the quench-flow analysis (), and fitting only a single kinetic parameter, the off-rate (), together with the fluorescence coefficients for substrate (), enzyme–substrate complex () and product (; )

Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations