The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.
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We thank the Swiss Light Source (SLS) and ALBA beamline staff for their support. This work was supported by the Ministerio de Economía y Competitividad of Spain (BFU2011-23815/BMC to G.M.), the Novo Nordisk Foundation (grant NNF14CC0001 to G.M.), the Fundación Ramón Areces (to G.M.), the Comunidad Autónoma de Madrid (CAM-S2010/BMD-2305 to G.M.), the European Union Marie Curie 'SMARTBREAKER' (2010-276953 to S.S.), the Ministerio de Educación of Spain (SB2010-0105 to S.S.) and the Ministerio de Economia y Competitividad of Spain (JCI-2011-09308 to R.M. and BIO2012-32868 and ERC-SimDNA to M.O.).
The authors declare no competing financial interests.
Integrated supplementary information
A) pH 8/65ºC and B) pH6/40ºC. First-order rate constants (k*) are plotted against I-DmoI initial concentrations ([I-DmoI]0). The correlation coefficients (R) corresponding to the curve fitting to the equation describing k* as a function of [I-DmoI]0 and the estimated kinetic parameters k*max and K M* are shown in the chart. One gel for each assay is presented as an example of the experimental data.
Supplementary Figure 2 Superposition of the active centers of I-DmoI in the presence of Mg2+ or Mn2+.
Each structure is colored according to the nature of the present divalent ions. A) The initial state of the reaction previously obtained in the presence of the non-catalytic metal Ca2+ (PDB Code 2VS7) is superimposed with the initial experimental state obtained in this manuscript. The rmsd (root mean square deviation) of the atoms in the active centre is 0.2 Å. B) Detailed view of the active centre comparing the conformation of the state 2. The DNA phosphodiester bonds are still intact and sites A and B are occupied with the metal, Mg2+ or Mn2+ depending on the soaking. The rmsd of the atoms in the active centre is 0.1 Å. C) A similar detailed view of the enzyme catalytic centre in state 7. This is the final state where both DNA strands are cleaved in the same way independently of the metal used in the experiment (Mg2+ or Mn2+), and the metal is found in sites A and B. The rmsd of the atoms in the active centre is 0.1 Å. The Mg2+ structures were solved at 2.20 and 2.35 Å resolution for states 2 and 7 respectively.
Supplementary Figure 3 PELE simulation results: interaction energies (kcal/mol) versus distance (Å) for the Mn2+ ions’ entrance along the channel formed in the I-DmoI–DNA interface.
A different colour is used for the interaction energies obtained for the independent 127 PELE trajectories. Distances are relative to reference positions (i.e. in PDB structures). Positions of binding sites A, B and C are highlighted with blue circles. Graphs correspond to: A-B) modelling of Mn2+ binding to site A when no ions are previously located in the active site; C-D), modelling of Mn2+ binding to site B when no ions are previously bound in the active site; E-F), modelling of Mn2+ binding to site B when there is an ion bound in site A and G-H), modelling of Mn2+ binding to site C when there are ions bound in sites A and B. The green and grey spheres in Figures B, D, F. H are used to show the reference positions and the places visited by the Mn2+ ions throughout the PELE ‘effective’ trajectories (.i.e. corresponding to simulations where the ions accessed the active site).
Supplementary Figure 4 PB calculations in the structure corresponding to the first stage of the catalytic process of I-DmoI.
A) Electrostatic potential visualization of the surface in complex I-DmoI/DNA. The arrow indicates the aperture of the channel in the protein/DNA interface where site A is located. The DNA double helix is highlighted in yellow. B) Calculated Molecular Interaction Potential (MIP) using Mn2+ as probe for the structure corresponding to the initial stage of catalysis (state 1). The black mesh corresponds to the calculation assuming that no divalent ions are bound to I-DmoI. The red mesh corresponds to the calculation assuming that Mn2+ ions were previously bound to sites A and B. The 100 points of highest interaction energy (electrostatic + Van der Waals) are depicted in each case. The Mn2+ atoms are represented as black spheres and the residues involved in their coordination are shown. Notice that site C is predicted to bind Mn2+ even when divalent ions are previously bound to sites A and B. Panels C and D depict the Potential Energy Surface (PES) at the QM(BP86/SVP)/MM(AMBER) level of theory corresponding to the movement of a divalent ion from site A or B to C. C) The divalent ion was modelled as Mg2+. The 90 atoms considered in the QM partition are highlighted. The starting structure in the calculations is depicted in D), where black and red arrows indicate the displacements modelled from site A to C or B to C, respectively. Note that only one ion was present in the calculations, which is initially bound to sites A or B. QM/MM calculations show that there would be an energy barrier of ~15-20 kcal/mol associated with the displacement of the ion from A or B to C. Therefore, if an ion initially binds to A or B it would be probably trapped there instead of moving to C.
A) The K120M mutation disturbs double strand cleavage allowing the accumulation of nicked plasmid. 2 nM of supercoiled plasmid DNA target was incubated with 100 nM of wild type I-DmoI or 50, 100 nM of I-DmoI K120M mutant at pH 8, 65 ºC for 30 min. B) Nicked oligonucleotides labeled with 6-FAM (C and NC) were used to determine which DNA strand is affected by the K120M mutation in I-DmoI. Oligonucleotide and protein at 750 nM were incubated at pH 8, 65 ºC for 30 min. The left panel of the gel displays NC and C substrates in the absence of enzyme determining the non-cleaved status of the probes. In the right panel of the gel NC and C were incubated with I-DmoI K120M showing that the mutant activity to digest the intact coding strand in target C is normal; however, the mutant activity was affected when the NC probe (containing the intact non-coding strand) was used in the assay.
Supplementary Figure 6 Potential energy surface at the QM(B3LYP/SVP) level, considering a direct nucleophilic attack of a hydroxyl ion on the target phosphate group.
The same model depicted in Supplementary Figure 7A was considered [RC: Reaction Coordinate (combination of interatomic distances considered to model the reaction of interest)]. After optimization of the initial structure, a penta-coordinated phosphorous intermediate (similar to the one obtained in first step of the mechanism depicted in Supplementary Figure 7) is obtained, later this intermediate easily evolves to the final hydrolytic products (reaction energy of -32.7 kcal/mol at the QM(B3LYP/6-311++G(d,p)//B3LYP/SVP) level of theory). However, the distance between the O3’ leaving atom and the phosphate group as well as the pattern of coordination of metals in sites B and C are inconsistent with the crystallographic structures corresponding to states 5 to 7 of the catalytic mechanism of I-DmoI.
Supplementary Figure 7 QM(B3LYP/SVP) calculations in a model of the I-DmoI active site, considering a proton transfer between the nucleophilic water molecule and the O3′ leaving group.
A) The model considered (110 atoms) was based on the structure corresponding to time 8h (state 3). B) Potential Energy Surface (PES) of a first step where the target phosphate group deprotonates the incoming water molecule and renders a penta-coordinated phosphorous intermediate. The associated energy barrier and reaction energy of this step at the QM(B3LYP/6-311++G(d,p)//B3LYP/SVP) level are 24.8 and 20.4 kcal/mol respectively. C) PES corresponding to the final nucleophilic attack of the resulting hydroxyl ion and the protonation of the O3’ leaving group. The reaction energy considering the final product and the reactants is 1.0 kcal/mol at the QM(B3LYP/6-311++G(d,p)//B3LYP/SVP) level of theory RC: Reaction Coordinate (combination of interatomic distances considered to model the reaction of interest). D) Superimposition of the structure corresponding to the last stage of the catalytic process (state 7) after replacing the water in site C by Mg2+ and the same structure optimized at level QM(B3LYP/SVP)/MM(AMBER). The pre-optimized structure is depicted in dark gray. Notice that E117 is not coordinating the ion in C in the optimized structure, which negatively impacts its binding affinity.
Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 1503 kb)
Detailed view of the conformational changes in the active site of I-DmoI during catalysis showing the 7 reactant states. The differences in metal occupancy during the course of the reaction are represented with different tones of grey. The color code is the same as in the main figures 1-4. (MOV 3587 kb)
View of the E117 conformational change promoting metal exit in site C. The differences in metal occupancy during the course of the reaction are represented with different tones of grey. The color code is the same as in the main figures 1-4. (MOV 1211 kb)
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Molina, R., Stella, S., Redondo, P. et al. Visualizing phosphodiester-bond hydrolysis by an endonuclease. Nat Struct Mol Biol 22, 65–72 (2015). https://doi.org/10.1038/nsmb.2932
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