Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.     

Reaction of c-Type Cytochromes with the Iron Hexacyanides: Mechanistic Implications

The reaction of c-cytochromes with iron hexacyanides is similar in mechanism to the interaction of cytochromes with their physiological oxidants and reductants in that the formation of complexes precedes electron transfer. Analysis of the kinetics of oxidation and reduction of a number of c-cytochromes by solving the simultaneous differential equations defining the mechanism is possible, and allows assignment of all six rate constants describing a minimum three-step mechanism [cyto(Fe(+3)) + Fe(+2) right harpoon over left harpoon cyto (Fe(+3)) - Fe(+2) right harpoon over left harpoon cyto(Fe(+2)) - Fe(+3) right harpoon over left harpoon cyto(Fe(+2)) + Fe(+3)]. We find that the usual steady-state approximations are not valid. Furthermore, the ratio of first-order rate constants for electron transfer was approximately 1.0, and no correlation was found between any of the six rate constants and the differences in oxidation-reduction potential of the iron-hexacyanides and different cytochromes c. However, it was found that the ratio of the rate constants for complex formation between ferricytochrome c and potassium ferrocyanide and ferrocytochrome c and potassium ferricyanide was proportional to the difference in oxidation-reduction potentials. Thus the minimum three-step mechanism given above accurately describes the observed kinetic data. However, this mechanism leads to a number of conceptual difficulties. Specifically, the mechanism requires that the collision complexes formed [cyto(Fe(+3)) - Fe(CN)(6) (-4) and cyto(Fe(+2)) - Fe(CN)(6) (-3)] have very different equilibrium constants, and further requires that formation of the collision complexes be accompanied by "chemistry" to make the intermediates isoenergetic. A more complex five-step mechanism which requires that the reactants [Fe(CN)(6) (-4) and ferricytochrome c or Fe(CN)(6) (-3) and ferrocytochrome c] form a collision complex followed by a first-order process before electron transfer, was found to yield results similar to those of the three-step mechanism. However, describing the formation of the collision complex in terms of a rapid equilibrium circumvents conceptual difficulties and leads to a physically reasonable mechanism. In this mechanism the reactants are in rapid equilibrium with the collision complexes and the rate constants for complex formation are controlled by diffusion and accessibility. The collision complexes then rearrange, possibly through conformational changes and/or solvent reorganization, to yield isoenergetic intermediates that can undergo rapid reversible electron transfer. The five-step mechanism can be described by the same rate constants obtained from the three-step mechanism with the appropriate adjustments to account for rapid equilibrium. This more complex analysis associates the oxidation-reduction potential of a particular cytochrome with the relative magnitude of the first-order conversion of the oxidant and reductant collision complexes to their respective intermediates. Thus the cytochromes c control their oxidation-reduction potential by chemical and/or structural alterations. This mechanism appears to be general in that it is consistent with the observed kinetics of 11 different cytochromes c from a wide variety of sources with a range of oxidation-reduction potentials.     

Enzyme kinetics: Inhibition equations derived from the inelastic collision hypothesis

The inelastic collision hypothesis of enzyme action has been proposed by G. Medwedew. This theory has been here extended to the cases of competitive and non-competitive inhibitors, which form enzyme-inhibitor complexes, and to the cases of competitive and non-competitive substrates. The resulting equations are discussed and contrasted to those derived from the classical enzyme-substrate hypothesis. The original formulation of Medwedew neglects the presence of the enzyme-substrate complex although the general theory admits the formation of this complex. The formulation has been revised and extended to include complex formation. The resulting equation is discussed in terms of the usual criteria used to evaluate the Michaelis-Menten-Briggs-Haldane equation. Taken in part from a thesis submitted by Robert Katzman to the faculty of the Department of Physiology, University of Chicago, in partial fulfillment of the requirements for the Degree of Master of Science.     

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.     

Thermodynamics of nucleotide binding to actomyosin V and VI: a positive heat capacity change accompanies strong ADP binding

We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25 degrees C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15 degrees C and occurs with a large, positive change in heat capacity (DeltaC(P) degrees ) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (DeltaC(P) degrees ) and entropy (DeltaS degrees ) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.     

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.     

Crystallographic and kinetic evidence of a collision complex formed during halide import in haloalkane dehalogenase.

Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions. The rate-limiting step in the reaction of DhlA is the release of the halide ion. The kinetics of halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments. At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site. To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure. The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import. Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations. This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site. Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme. The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport.     

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.     

The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport

This review focuses on our studies over the past ten years which reveal that the mitochondrial inner membrane is a fluid-state rather than a solid-state membrane and that all membrane proteins and redox components which catalyze electron transport and ATP synthesis are in constant and independent diffusional motion. The studies reviewed represent the experimental basis for therandom collision model of electron transport. We present five fundamental postulates upon which the random collision model of mitochondrial electron transport is founded: (1) All redox components areindependent lateral diffusants; (2) Cytochromec diffuses primarily inthree dimensions; (3) Electron transport is adiffusion-coupled kinetic process; (4) Electron transport is amulticollisional, obstructed, long-range diffusional process; (5) The rates of diffusion of the redox components have a direct influence on the overall kinetic process of electron transport and can berate limiting, as indiffusion control. The experimental rationales and the results obtained in testing each of the five postulates of the random collision model are presented. In addition, we offer the basic concepts, criteria and experimental strategies that we believe are essential in considering the significance of the relationship between diffusion and electron transport. Finally, we critically explore and assess other contemporary studies on the diffusion of inner membrane components related to electron transport including studies on: rotational diffusion, immobile fractions, complex formation, dynamic aggregates, and rates of diffusion. Review of all available data confirms the random collision model and no data appear to exist that contravene it. It is concluded that mitochondrial electron transport is a diffusion-based random collision process and that diffusion has an integral and controlling affect on electron transport.     

Contributions of collision rate and collision efficiency to erythrocyte aggregation in postcapillary venules at low flow rates

Red blood cell aggregation at low flow rates increases venous vascular resistance, but the process of aggregate formation in these vessels is not well understood. We previously reported that aggregate formation in postcapillary venules of the rat spinotrapezius muscle mainly occurs in a middle region between 15 and 30 microm downstream from the entrance. In light of the findings in that study, the main purpose of this study was to test two hypotheses by measuring collision frequency along the length of the venules during low flow. We tested the hypothesis that aggregation rarely occurs in the initial 15-microm region of the venule because collision frequency is very low. We found that collision frequency was lower than in other regions, but collision efficiency (the ratio of aggregate formation to collisions) was almost nil in this region, most likely because of entrance effects and time required for aggregation. Radial migration of red blood cells and Dextran 500 had no effect on collision frequency. We also tested the hypothesis that aggregation was reduced in the distal venule region because of the low aggregability of remaining nonaggregated cells. Our findings support this hypothesis, since a simple model based on the ratio of aggregatable to nonaggregatable red blood cells predicts the time course of collision efficiency in this region. Collision efficiency averaged 18% overall but varied from 0 to 52% and was highest in the middle region. We conclude that while collision frequency influences red blood cell aggregate formation in postcapillary venules, collision efficiency is more important.     

The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex

Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.     

Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.

The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.     

Chirally sensitive collision induced dissociation of proton‐bound diastereomeric complexes of tryptophan and 2‐butanol

In this work we report the stereo‐dependent collision‐induced dissociation (CID) of proton‐bound complexes of tryptophan and 2‐butanol. The dissociation efficiency was measured as a function of collision energy in single collision mode. The homochiral complex was found to be less stable against CID than a heterochiral one. Additional gas dependence measurements were performed with diastereomeric complexes that confirm the findings.     

Peculiar 2-aminopurine fluorescence monitors the dynamics of open complex formation by bacteriophage T7 RNA polymerase

The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm. This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12. The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1). Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1).     

Effect of Dextran 500 on Radial Migration of Erythrocytes in Postcapillary Venules at Low Flow Rates

Recently, we reported that collision efficiency (fraction of total collisions that result in the formation of aggregates) between red blood cells was an important factor in the formation of aggregates in postcapillary venules. In the present study, we focus on how high molecular weight dextran influences the overall radial migration trend of red blood cells in the postcapillary venule along a longitudinal distance of 50 μm from the bifurcation which would in turn affect collision behavior of these cells. A radial migration index, which defines the extent of radial migration of individual cells relative to the vessel center, was found to have a larger magnitude after infusion of dextran (1.9 ± 2.73) compared to that before dextran infusion (1.48 ± 3.89). This implied that dextran-induced aggregation might provide an external force to actively move cells towards the centerline of the vessel, which could contribute to the greater number of red blood cells participating in collision (16% increase) and aggregate formation. Further analysis of the collision behavior of individual red blood cells revealed that collision frequencies of individual cells decreased from a wide range (1 to 14) to a narrow range (1 to 5) after dextran treatment, indicating the alteration of collision behavior of red blood cells by the presence of aggregates along the flow stream.     

Platelet adhesive dynamics. Part I: characterization of platelet hydrodynamic collisions and wall effects

Abnormally high shear stresses encountered in vivo induce spontaneous activation of blood platelets and formation of aggregates, even in the absence of vascular injury. A three-dimensional multiscale computational model—platelet adhesive dynamics—is developed and applied in Part I and Part II articles to elucidate key biophysical aspects of GPIbα-von-Willebrand-factor-mediated interplatelet binding that characterizes the onset of shear-induced platelet aggregation. In this article, the hydrodynamic effects of the oblate spheroidal shape of platelets and proximity of a plane wall on the nature of cell-cell collisions are systematically investigated. Physical quantities characterizing the adhesion probabilities between colliding platelet surfaces for the entire range of near-wall encounters between two platelets are obtained for application in platelet adhesive dynamics simulations of platelet aggregation explored in a companion article. The technique for matching simulation predictions of interplatelet binding efficiency to experimentally determined efficiencies is also described. Platelet collision behavior is found to be strikingly different from that of spheres, both close to and far from a bounding wall. Our results convey the significant effects that particle shape and presence of a bounding wall have on the particle trajectories and collision mechanisms, collision characteristics such as collision time and contact area, and collision frequency.     

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.     

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp.     

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organisation of repair

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the "random collision" model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a "processive" mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. These two models give different predictions concerning the time course of repair. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate of repair until scanning is complete. We have examined the time course of DNA repair in human fibroblasts given low (generally sublethal) doses of UV light. Using 3 distinct assays, we find no sign of a constant repair rate after 4 J/m2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. The implications of this finding for the structural organisation of repair are discussed.