A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping

Uracil DNA glycosylase (UNG) is the primary enzyme for the removal of uracil from the genome of many organisms. A key question is how the enzyme is able to scan large quantities of DNA in search of aberrant uracil residues. Central to this is the mechanism by which it flips the target nucleotide out of the DNA helix and into the enzyme-active site. Both active and passive mechanisms have been proposed. Here, we report a rapid kinetic analysis using two fluorescent chromophores to temporally resolve DNA binding and base-flipping with DNA substrates of different sequences. This study demonstrates the importance of the protein–DNA interface in the search process and indicates an active mechanism by which UNG glycosylase searches for uracil residues

A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (kcat/Km) compared with the viral enzyme due to a lower Km. The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2′-deoxypseudouridine and 2′-α-fluoro-2′-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U·A and U·G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair

One recognition sequence, seven restriction enzymes, five reaction mechanisms

The diversity of reaction mechanisms employed by Type II restriction enzymes was investigated by analysing the reactions of seven endonucleases at the same DNA sequence. NarI, KasI, Mly113I, SfoI, EgeI, EheI and BbeI cleave DNA at several different positions in the sequence 5′-GGCGCC-3′. Their reactions on plasmids with one or two copies of this sequence revealed five distinct mechanisms. These differ in terms of the number of sites the enzyme binds, and the number of phosphodiester bonds cleaved per turnover. NarI binds two sites, but cleaves only one bond per DNA-binding event. KasI also cuts only one bond per turnover but acts at individual sites, preferring intact to nicked sites. Mly113I cuts both strands of its recognition sites, but shows full activity only when bound to two sites, which are then cleaved concertedly. SfoI, EgeI and EheI cut both strands at individual sites, in the manner historically considered as normal for Type II enzymes. Finally, BbeI displays an absolute requirement for two sites in close physical proximity, which are cleaved concertedly. The range of reaction mechanisms for restriction enzymes is thus larger than commonly imagined, as is the number of enzymes needing two recognition sites

One recognition sequence, seven restriction enzymes, five reaction mechanisms

The diversity of reaction mechanisms employed by Type II restriction enzymes was investigated by analysing the reactions of seven endonucleases at the same DNA sequence. NarI, KasI, Mly113I, SfoI, EgeI, EheI and BbeI cleave DNA at several different positions in the sequence 5′-GGCGCC-3′. Their reactions on plasmids with one or two copies of this sequence revealed five distinct mechanisms. These differ in terms of the number of sites the enzyme binds, and the number of phosphodiester bonds cleaved per turnover. NarI binds two sites, but cleaves only one bond per DNA-binding event. KasI also cuts only one bond per turnover but acts at individual sites, preferring intact to nicked sites. Mly113I cuts both strands of its recognition sites, but shows full activity only when bound to two sites, which are then cleaved concertedly. SfoI, EgeI and EheI cut both strands at individual sites, in the manner historically considered as normal for Type II enzymes. Finally, BbeI displays an absolute requirement for two sites in close physical proximity, which are cleaved concertedly. The range of reaction mechanisms for restriction enzymes is thus larger than commonly imagined, as is the number of enzymes needing two recognition sites

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 (; )

A Switch in the Mechanism of Communication between the Two DNA-Binding Sites in the SfiI Restriction Endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer