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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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B) The closed β2Hβ conformation in the mTdp2–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Wall-eyed stereo view is displayed. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2 (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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mTdp2 WT (lanes 1–13) or mTdp2 D358N (lanes 14–26) were incubated in the presence or absence of Mg and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl of trypsin.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Experiments performed as in panel D for mTdp2 WT (lanes 27–39) or mTdp2 D358N (lanes 40–52), but with chymotrypsin instead of trypsin.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg occupancy and substrate binding loops observed for the individual protomers.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg coordinating residue Asp358.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg binding in the Tdp2 active site.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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To evaluate Mg and DNA-dependent Tdp2 structural states in solution, we probed mTdp2 conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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In the absence of DNA or Mg, mTdp2 is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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By comparison, Mg, and to a greater extent Mg/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2 from proteolytic cleavage.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Interestingly, addition of Mg alone protects against proteolysis as well.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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This is consistent with Mg stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2 domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Comparisons of the human hTdp2-DNA complex structure to the mTdp2 DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Moreover, similar to mTdp2, proteolytic protection of the hTdp2 substrate binding loop occurs with addition of Mg and DNA (Supplementary Figure S3B).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Consistently in high-resolution X-ray structural analyses we, (8,20) and others (36) observe a single Mg metal bound in the Tdp2 active site.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction (37).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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In these experiments, at limiting Mg concentrations, Ca addition to Tdp2 reactions stimulated activity.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site (38).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center (20), and we propose this might account for varied results in different studies.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn and Ca), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Metal cofactor interactions with Tdp2. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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A) Intrinsic tryptophan fluorescence of mTdp2 was used to monitor a conformational response to divalent metal ion binding.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Either Mg or Ca were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Both Mg and Ca induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2 in the presence and absence of DNA, while D358N active site mutant of mTdp2 is unresponsive to Mg. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
B) mTdp2 activity assayed on a T5PNP substrate as a function of Mg and Ca concentration.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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PNP release (monitored by absorbance at 415 nm) as a function of Mg concentration and in the absence or presence of 1 or 10 mM Ca is shown; error bars, s.d.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
n = 4. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2–DNA–Mn complex (PDB entry 5INP) show a single Mn (cyan) is bound with expected octahedral coordination geometry.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn as the identity of this atom. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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D) Comparison of Ca (green Ca ion, orange DNA) (PDB entry 5INQ), and Mg (magenta Mg ion, yellow DNA) (PDB entry 4GZ1) mTdp2–DNA structures shows that Ca distorts the 5′-phosphate binding mode.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Our proteolysis results indicate a Mg-dependent Tdp2 conformational response to metal binding.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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This analysis revealed Mg Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2 (20,39).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Inclusion of ultrapure Ca (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg (Figure 4B).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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We performed the same titrations with human hTdp2 and hTdp2 (Supplementary Figure S4), and find similar stimulation of activity by Mg and inhibition by Ca.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Overall, these metal binding analyses are consistent with a single metal ion mediated reaction.
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn) (20) or inhibit (Ca, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Anomalous difference Fourier maps of the Tdp2–DNA–Mn complex show a single binding site for Mn in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn ligands (Supplementary Table S3).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
The Mn ion is positioned in the Tdp2 active site similar to the Mg-bound complex (Figure 2C), which is consistent with the ability of Mn to support robust Tdp2 catalytic activity (20).
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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In contrast, while co-complex structures with Ca also show a single metal ion, Ca binds in a slightly different position, shifted ∼1 Å from the Mg site.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Although Ca is also octahedrally coordinated, longer bond lengths for the Ca ligands (Supplementary Table S3) shift the Ca ion relative to the Mg ion site.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Interestingly, bi-dentate inner sphere metal contacts from the Ca ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Together with results showing that under the conditions examined here, Ca inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca-mediated inhibition of the Tdp2 reaction.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Next, to examine the feasibility of our proposed single Mg mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus (20).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Structure-function analysis of the Tdp2 reaction mechanism. (
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PMC4857006
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Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Residue numbers shown are for the mTdp2 homolog. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Reaction proceeds from right to left. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
C) Models for the mTdp2-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Residue numbers shown are for the mTdp2 homolog. (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2 fusion proteins on the three substrates.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2; error bars, s.d.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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n = 3.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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A starting model was generated from atomic coordinates of the mTdp2 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2 5′-aminohexanol substrate analog structure (3.2–3.4 Å).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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No appreciable energy penalty is observed during the first 0.5 Å of the reaction coordinate.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
When the reaction reaches an O–P distance of 2.18 Å, formation of a transition state with an energy maximum of +7.4 kcal mol is observed.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
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Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Concomitant with this, the phosphotyrosyl O–P bond weakens (d = 1.89 Å), and the formation of the penta-covalent transition state (Figure 5C ‘ii-transition state’) is observed.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0 (40).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
In the final optimized structure, the observed product state (Figure 5C, ‘iii-product’) is found in a conformation that is 7.4 kcal mol more stable than the initial reactive state (Figure 5B).
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
The tyrosine oxy-anion product is coordinated to the Mg ion with a 2.0 Å distance, which is the shortest of the six Mg ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg greatly stabilizes the product oxy-anion.
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PMC4857006
|
Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.
|
An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group.
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