Dissecting protein-induced DNA looping dynamics in real time.

Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA-protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA-protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA-protein dynamics in real time.

Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping.

Many enzymes acting on DNA require Mg(2+) ions not only for catalysis but also to bind DNA. Binding studies often employ Ca(2+) as a substitute for Mg(2+), to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca(2+) mimics Mg(2+), Ca(2+) causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg(2+) present as the DNA is cleaved so, to study the effect of Mg(2+) on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca(2+) or Mg(2+) but, unlike wild-type SfiI with Ca(2+), the binding was reversible. With both mutants, dissociation was slow with Ca(2+) but was in one case much faster with Mg(2+). Hence, Ca(2+) can affect DNA binding differently from Mg(2+). Moreover, SfiI is an archetypal system for DNA looping; on DNA with two recognition sites, it binds to both sites and loops out the intervening DNA. While the dynamics of looping cannot be measured with wild-type SfiI and Ca(2+), it becomes accessible with the mutant and Mg(2+).

Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands.

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting 'top' and 'bottom' strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.

Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites.

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.

Dynamics and consequences of DNA looping by the FokI restriction endonuclease.

Genetic events often require proteins to be activated by interacting with two DNA sites, trapping the intervening DNA in a loop. While much is known about looping equilibria, only a few studies have examined DNA-looping dynamics experimentally. The restriction enzymes that cut DNA after interacting with two recognition sites, such as FokI, can be used to exemplify looping reactions. The reaction pathway for FokI on a supercoiled DNA with two sites was dissected by fast kinetics to reveal, in turn: the initial binding of a protein monomer to each site; the protein-protein association to form the dimer, trapping the loop; the subsequent phosphodiester hydrolysis step. The DNA motion that juxtaposes the sites ought on the basis of Brownian dynamics to take approximately 2 ms, but loop capture by FokI took 230 ms. Hence, DNA looping by FokI is rate limited by protein association rather than DNA dynamics. The FokI endonuclease also illustrated activation by looping: it cut looped DNA 400 times faster than unlooped DNA.

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.

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.

The asymmetric ATPase cycle of the thermosome: elucidation of the binding, hydrolysis and product-release steps.

Using a combination of intrinsic fluorescence to report ATP-induced rearrangements, quenched-flow to measure ATP hydrolysis "on-enzyme" and optical methods to probe the kinetics of product release, we have begun to dissect the process of energy transduction in the thermosome, a type II chaperonin from Thermoplasma acidophilum. Stoichiometric measurements of ATP binding reveal the tight association of eight nucleotide molecules per hexa-decamer, implying the filling of only one ring owing to strong negative cooperativity. After binding, we show that these eight ATP molecules are hydrolysed over the next 50 s, after which hydrolysis slows down markedly during the establishment of the steady state in the ATPase reaction, demonstrating that the kinetic system is off-rate limited. Looking in more detail, this rapid first-turnover can be dissected into two phases; the first occurring with a half-time of 0.8 s, the second with a half-time of 14 s, possibly reflecting the differential behaviour of the four alpha and four beta subunits in a single thermosome ring. To investigate the post-hydrolytic events, we used two heat-stable enzyme-linked optical assays to measure the rate of evolution of ADP and of phosphate from the thermosome active site. Neither product showed a rapid dissociation phase prior to the establishment of the steady state, showing that both are released slowly at a rate that limits the cycle. These data highlight the importance of the highly populated thermosome/ADP/Pi complex in the molecular mechanism.

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 (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). 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'-alpha-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.

Cleavage of individual DNA strands by the different subunits of the heterodimeric restriction endonuclease BbvCI.

BbvCI cleaves an asymmetric DNA sequence, 5'-CC downward arrow TCAGC-3'/5'-GC downward arrow TGAGG-3', as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R(1), which acts at GC downward arrow TGAGG; and R(2), which acts at CC downward arrow TCAGC. Some mutants of BbvCI with defects in one subunit, either R(1)(-)R(2)(+) or R(1)(+)R(2)(-), cleave only one strand, that attacked by the native subunit. In analytical ultracentrifugation at various concentrations of protein, wild-type and mutant BbvCI enzymes aggregated extensively, but are R(1)R(2) heterodimers at the concentrations used in DNA cleavage reactions. On a plasmid with one recognition site, wild-type BbvCI cleaved both strands before dissociating from the DNA, while the R(1)(-)R(2)(+) and R(1)(+)R(2)(-) mutants acted almost exclusively on their specified strands, albeit at relatively slow rates. During the wild-type reaction, the DNA is cleaved initially in one strand, mainly that targeted by the R(1) subunit. The other strand is then cleaved slowly by R(2) before the enzyme dissociates from the DNA. Hence, the nicked form accumulates as a transient intermediate. This behaviour differs from that of many other restriction enzymes, which cut both strands at equal rates. However, the activities of the R(1)(+) and R(2)(+) subunits in the wild-type enzyme can differ from their activities in the R(1)(+)R(2)(-) and R(1)(-)R(2)(+) mutants. Each active site in BbvCI therefore influences the other.

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.