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Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.


INTRODUCTION
Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling--over-and under-winding of DNA strands, respectively--and the linking together (catenation) of DNA strands. Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. The mammalian type II topoisomerases Top2␣ and Top2␤ enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions (1)(2)(3). Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5 -phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).
The Top2cc protein-DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries (4)(5)(6)(7). To promote cancer cell death, Top2 reactions are 'poisoned' by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides (8,9), abasic sites (10)(11)(12)(13)(14) and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (15,16) ( Figure 1A). In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5linked Top2 DNA-protein crosslink. The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored.
Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5 -phosphotyrosyl (5 -Y) linkages, and is a key Figure 1. Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5 -Top2 protein adduct linked by a phosphotyrosine bond. Tdp2 hydrolyzes the 5 -phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5 -phosphate, which facilitates DNA end joining through the NHEJ pathway. (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (⑀A) or an abasic site (THF) and a 5 -nitrophenol moiety. Phosphotyrosyl bond hydrolysis catalyzed by mTdp2 cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2 cat reaction rates on p-nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s −1 produced by mTdp2 cat . P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2 cat bound to 5 -phosphate DNA (product complex) containing ⑀A (yellow). DNA binding ␤2H␤-grasp (tan) and cap elements engage the 5 -nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2 cat bound to 5 -phosphate DNA (product complex) containing THF (yellow). DNA binding ␤2H␤-grasp (tan) and cap elements engage the 5 -nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2 cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. PDB entry 5INM is displayed, also see Table 1. modulator of cellular resistance to chemotherapeutic Top2 poisons (17)(18)(19)(20). Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear ␥ H2AX foci, a marker of DSBs (17), underlining the importance of Tdp2 in cellular Top2cc repair. Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression (21). The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating muta-tions are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2␤-dependent transcription (18). It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood.
Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2 cat ) bound to a DNA substrate mimic, and a 5 -phosphorylated reaction product (20). However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain. First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. Based on metal-bound Tdp2 structures (20), we also proposed a single Mg 2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N 6 -etheno-adenine) DNA damage. Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a singlemetal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.

Generation of PNP modified oligonucleotide substrates
Oligonucleotides with a 5 -phosphate modification were obtained from IDT and diluted to a concentration of 2 mmol l −1 in water. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; Pierce) was used to couple p-nitrophenol to the oligonucleotide with a modified version of the manufacturer's instructions. Briefly, 25 mg EDC were dissolved in 150 l oligonucleotide solution. After the EDC dissolved, 400 l DDW, 60 l 100 mmol l −1 Imidazole pH 6 and 100 l of PNP from a saturated water/PNP mixture preheated to 55 • C were added. Reactions were heated at 55 • C for 30 min to ensure the PNP dissolved, then incubated at 37 • C overnight. Reactions were quenched with 500 l 2 mol l −1 acetic acid and heated to 55 • C for 1 h, followed by neutralization with 500 l 2 mol l −1 Tris base. Reactions were twice diluted to 15 ml in DDW then concentrated to 300 l with a 3K MWT cutoff spin concentrator (Amicon), then run on 20% (w/v) 19:1 8M urea-TBE PAGE to resolve reaction products. Bands were visualized by UV shadow, excised, soaked in 15 ml DDW for 16 h at 15 • C, and purified on a C18 reverse-phase Sep-Pac (Waters).
X-ray diffraction data collection, phasing and refinement X-ray data (Table 1) for all structures except the manganese soak (PDB entry 5INP) were collected at 100 K on beamline 22-ID of the Advanced Photon Source at a wavelength of 1.000Å. X-ray data for the manganese soak (PDB entry 5INP) were collected on a Rigaku HF007 Cu rotating anode X-ray source at a wavelength of 1.5418Å. X-ray diffraction data were processed and scaled using the HKL2000 suite (22). The hTdp2 cat -DNA and mTdp2 cat -apo crystals were phased by molecular replacement in PHASER (23) using chain A of PDB entry 4GZ1. Initial solutions were improved by iterative rounds of manual fitting in COOT (24) and refinement in PHENIX (25).

Limited proteolysis assays
For proteolysis experiments, 4 l reactions containing 40 mol l −1 mTdp2 cat (aa 118 to 370) in reaction buffer (10 mmol l −1 HEPES pH 7.5, 200 mmol l −1 NaCl, 0.5 mmol L −1 TCEP) with 4 mmol l −1 Mg(OAc) 2 (indicated +Mg) or 8 mmol l −1 NaOAc (indicated -Mg), with 0 or 60 mol l −1 12 nt DNA were incubated with 0, 5, 1.7 or 0.6 g l −1 Trypsin for 1 h at 22 • C. Reactions were quenched by addition of Laemmli SDS-PAGE dye, heated at 70 • C for 10 min, and analyzed by SDS-PAGE. For mass-spectrometry analysis of peptide masses, reactions were quenched with 1% (v/v) trifluoroacetic acid , purified on a C18 ZipTip (Millipore) and an ESI-MS mass measurement made on a Q-ToF Ultima/Global (Micromass/Waters) using flow injection from a pressurized bomb. The instrument was operated in the positive ion, V-mode and calibrated using the multiply-charged ion envelope of horse heart cytochrome C. The molecular ion mass was determined using the Max Ent 1 routine from the MassLynx software.

Preparation of NHEJ substrates
Oligonucleotide 300 bp substrates with 5 phosphorylated GATC overhangs were generated as previously described (26). Comparable substrate with 5 -phophotyrosine adducted GATC overhangs were generated by annealing the modified strand to complementary strands that generate caps for substrate head and tail ends. Head and tail caps have a 5 -phosphotyrosine-GATC overhang terminus on one end; the 5 -phosphorylated, non-adducted overhangs on the other end are made complementary to the head and tail ends of a 270 bp double stranded core fragment generated by polymerase chain reaction. Ligation of an excess of these caps to the 270 bp fragment generates a 300 bp substrate with 5 -phosphotyrosine end structures as described in Figure 7. Unligated caps are removed using a Qiaquick PCR cleanup kit.

In vitro NHEJ reactions
Purified NHEJ proteins (Ku, XRCC4-ligase IV, XLF) were prepared as previously described (27). End joining reactions were performed using 5 nM DNA substrate, 25  Reactions were stopped by the addition of 0.1% (w/v) SDS and 5 mM EDTA and analyzed by 5% native PAGE.

Cellular NHEJ assays
Mouse embryo fibroblast (MEF) cells from matched Tdp2 +/+ and Tdp2 −/− mice (19) were a gift from F. Cortes-Ledesma, and were immortalized by transformation with a construct that expresses SV40 T-antigen (Addgene #1779). HCT-116 cells and a ligase IV deficient variant were the gift of E. A. Hendrickson (28). The cDNAs with wild-type human Tdp2 and polymorphic variants generated by mutagenesis were introduced into pLX302 (29) (Addgene#25896) to prepare lentivirus. Tdp2 −/− cells were infected with lentivirus and bulk cell cultures expressing lentiviral delivered construct purified by treatment with puromycin. Expression of human Tdp2 (hTdp2 FL ) was validated by Western analysis (12203-1-AP; Proteintech). Fifty nanograms of the 300 bp substrates used in in vitro experiments and 2 g of carrier supercoiled plasmid DNA were introduced into 2 × 10 5 MEF or HCT 116 cells by electroporation (Neon, Invitrogen) using a 10 l chamber and a single 1350 V, 30 ms pulse. Cells were recovered 1 h later, washed with phosphate buffered saline and DNA purified using a Qiamp DNA mini kit on a Qiacube. NHEJ products were quantified by qPCR and characterized by amplicon sequencing as previously described (27).

Etoposide Clonogenic survival assays
Clonogenic survival assays were carried out by treating log phase cells with Etoposide as described in the legend to Figure 7 before seeding treated and mock treated cells in 10 cm dishes. Colonies formed after 10 days post-treatment were strained using a crystal violet (0.5% w/v) solution. Plates containing a minimum of 50 colonies were counted by hand, and at least three plates were counted for each dose.

QM/MM
In the QM/MM calculation, in addition to the water nucleophile and the putative catalytic Lewis base (Asp272), the side chains of proximal residues (Asn130, Asn274, His236, His326, His359, Ser239 and Glu162), the bound phosphate moiety, the Mg 2+ and three waters in its coordinate shell are all included in the QM sub-system. In addition, we modeled the position of the Top2 peptide tyrosine based on conformations of the substrate analog. The QM sub-system of this study consisted of 110 atoms with a zero net charge on the sub-system. QM/MM calculations are performed using Gaussian09.D01 (30). Since the QM sub-system contains a large number of buried atoms and commonly used electrostatic potential fitting schemes to obtain the charge distribution at atomic positions become unreliable for such systems with buried atoms, we have selected the CM5 charge model in the current study to calculate the charges on atomic positions at each step of the QM/MM calculation. This CM5 scheme is an extension to the Hirshfeld population analysis and is adapted to handle buried charges properly. This charge distribution of the QM sub-system was used to evaluate contributions of residues in the MM region to the net stability of the transition and product states as compared to that of the initial reactive state. As reported previously (31), this residue analysis is solely based on the electrostatic energy contributions to the initial, transition and product states. According to this estimation, the residues Arg142, Lys213, Ser235, Asp277, Glu279, Asp292, Glu295, Asp308, Asp343, Arg354 and Trp360 contribute toward the stability of both the transition and product states as compared with the reactant. However, the electrostatic contributions from residues Asp132, Asp135, Glu186, Glu242, Arg247, Thr273, Arg276, Lys299, Lys322, Arg324, Arg327 and Asp358 have the opposite effect toward the stabilities of the transition and product states.

Tdp2 processing of compound DNA damage
Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N 6ethenoadenine, ⑀A) (16) and abundant abasic sites (13) ( Figure 1A). Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known. To test this, we adapted an EDC coupling method to generate 5 -terminal p-nitrophenol (PNP) modified oligonucleotides (32,33) that also harbored DNA damage at the 5 -nucleotide position (see Materials and Methods). We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2 cat ) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5 -terminus of compound damaged DNA substrates using a colorimetric assay ( Figure 1B). We observe robust Tdp2-dependent release of PNP from 5 -modified oligonucleotides in the context of dA-PNP, ⑀A-PNP or the abasic-site analog tetrahydrofuran spacer (THF) ( Figure  1C). Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5 lesions composed of abasic or bulky DNA base adduct DNA damage.
To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2 cat bound to 5 -phosphate DNA (product complex) with a 5 -⑀A at 1.43 A resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5 -THF at 2.15Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). In these Tdp2-DNA complex structures, mTdp2 cat adopts a mixed ␣-␤ fold typified by a central 12-stranded anti-parallel ␤-sandwich enveloped by several helical elements that mold the Tdp2 active site. One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate. In the DNA lesionbound state, two key DNA binding elements, the ␤-2-helix-␤ (␤2H␤) 'grasp', and 'helical cap' mold the substrate binding trench and direct the ssDNA of a 5 -overhang substrate into the active site. A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.
The mode of engagement of the 5 -nucleobase of the bulky ⑀A adduct describes a mechanism for Tdp2 to bind 5 -tyrosylated substrates that contain diverse forms of DNA damage. The 5 -⑀A nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the 'hydrophobic wall' that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).
For comparison, we also determined a structure of an undamaged 5 -adenine (5 -dA) bound to Tdp2 at 1.55Å (PDB entry 5INL). A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ⑀A, dA and dC, Supplementary Figure S1A-D). Therefore, structurally diverse undamaged or alkylated bases (e.g. ⑀G, ⑀T) (34,35) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the ␤2H␤ motif. Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5terminus ( Figure 2C). Interestingly, in the absence of a nucleobase, O4 of the THF ring adopts a close approach (2.8 A) to a water molecule that directly participates in the outer sphere single Mg 2+ ion coordination shell ( Figure 2D). This shift is coincident with a small adjustment in the position of the +2 and +3 nucleotides (Supplementary Figure S1E). These collective differences may explain the slight, but statistically significant elevated activity on the THF substrate ( Figure 1C).

Structural plasticity in the Tdp2 DNA binding trench
An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein-water-protein contacts that span a gap between the catalytic core and the DNA binding ␤2H␤-grasp (Supplementary Figure S2). In this arrangement, six solvent molecules form a channel under the ␤2H␤-grasp, ending with hydrogen bonds to the peptide backbone of the Mg 2+ ligand Asp358. The paucity of hydrophobic interactions stabilizing the ␤2H␤ DNA-bound conformation suggests that conformational plasticity in the ␤2H␤ might be a feature of DNA damage and metal cofactor engagement. To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4Å resolution (PDB entry 5INM; Figures 1F and 3A).
This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg 2+ occupancy and substrate binding loops observed for the individual protomers. The most striking feature of the DNA ligand-free state is that the active site ␤2H␤-grasp can adopt alternative structures that are distinct from the DNA-bound, closed ␤2H␤ DNA binding grasp ( Figure 3A and B). In one monomer (chain 'E'), the grasp adopts an 'open' 3-helix loop conformation that projects away from the EEP catalytic core. Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. The remaining two molecules in the DNA-free crystal form are closed ␤2H␤ conformers similar to the DNA bound structures ( Figure 3C). Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the ␤2H␤ grasp, rather than inducing closure upon binding.
A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. 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. 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 ␤2H␤ fold. By comparison, the closed ␤2H␤ grasp conformer is stabilized by Asn312 and Asn314 binding into two ␤2H␤ docking pockets, and Leu315 engagement of the 5 -terminal nucleobase ( Figure 3B). To transition into the closed ␤2H␤ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the ␤2H␤ antiparallel ␤-sheet. Stabilization of the closed ␤2H␤-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg 2+ coordinating residue Asp358. Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg 2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the ␤2H␤ DNA binding grasp, and these rearrangements are coordinated with Mg 2+ binding in the Tdp2 active site.
To evaluate Mg 2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2 cat conformations using limited trypsin and chymotrypsin proteolysis ( Figure 3C-E). In the absence of DNA or Mg 2+ , mTdp2 cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). By comparison, Mg 2+ , and to a greater extent Mg 2+ /DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2 cat from proteolytic cleavage. Interestingly, addition of Mg 2+ alone protects against proteolysis as well. This is consistent with Mg 2+ stabilizing the closed conformation of the ␤2H␤-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the ␤2H␤-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).
To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2Å resolution structure of the human Tdp2 cat domain bound to a DNA 5 -PO 4 terminus product complex (PDB entry 5INO). Comparisons of the human hTdp2 cat -DNA complex structure to the mTdp2 cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). Moreover, similar to mTdp2 cat , proteolytic protection of the hTdp2 cat substrate binding loop occurs with addition of Mg 2+ and DNA (Supplementary Figure S3B). Thus, Xray structures and limited proteolysis analysis indicate that DNA-and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. (C) Structure of mTdp2 cat bound to 5 -phosphate DNA (product complex) containing THF (yellow), Mg 2+ (magenta) and its inner-sphere waters (gray). mTdp2 cat is colored with red (electronegative), blue (electropositive) and gray (hydrophobic) electrostatic surface potential displayed. PDB entry 5INK is displayed. (D) -A weighted 2Fo-Fc electron density map (at 2.15Å resolution, contoured at 2.0 ) for THF-DNA complex. The THF is shown in yellow and a hydrogen bond from the THF O4 to inner-sphere water is shown as gray dashes.

Tdp2 metal ion dependence
Consistently in high-resolution X-ray structural analyses we, (8,20) and others (36) observe a single Mg 2+ metal bound in the Tdp2 active site. 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). However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction (37). In these experiments, at limiting Mg 2+ concentrations, Ca 2+ addition to Tdp2 reactions stimulated activity. 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). 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. 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 2+ and Ca 2+ ), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).
Our proteolysis results indicate a Mg 2+ -dependent Tdp2 conformational response to metal binding. The Tdp2 active site has three tryptophan residues within 10Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2 cat . These data were an excellent fit to a singlesite binding model both in the presence and absence of DNA ( Figure 4A). This analysis revealed Mg 2+ K d 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). We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5 -phosphate by mTdp2 cat (20,39). This small molecule substrate is not expected to be influenced by metal-DNA coordination outside of the active site. Inclusion of ultrapure Ca 2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting monomer E of the DNA-free mTdp2 cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. The ␤2H␤ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. Wall-eyed stereo view is displayed. (B) The closed ␤2H␤ conformation in the mTdp2 cat -DNA product structure containing 5 -⑀A (yellow, PDB entry 5HT2). T309 (green) is an integral part of the ␤2H␤ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the ␤2H␤ docking pocket. Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2 cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as '∼' (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. mTdp2 cat WT (lanes 1-13) or mTdp2 cat D358N (lanes [14][15][16][17][18][19][20][21][22][23][24][25][26] were incubated in the presence or absence of Mg 2+ and/or a 12 nt self annealing, 5 -phosphorylated DNA (substrate '12 nt' in Supplementary Table S1), then reacted with 0.6, 1.  Mg 2+ ( Figure 4B). We performed the same titrations with human hTdp2 FL and hTdp2 cat (Supplementary Figure S4), and find similar stimulation of activity by Mg 2+ and inhibition by Ca 2+ . Overall, these metal binding analyses are consistent with a single metal ion mediated reaction.
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 2+ ) (20) or inhibit (Ca 2+ , Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). Anomalous difference Fourier maps of the Tdp2-DNA-Mn 2+ complex show a single binding site for Mn 2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn 2+ ligands (Supplementary Table S3). The Mn 2+ ion is positioned in the Tdp2 active site similar to the Mg 2+ -bound complex ( Figure 2C), which is consistent with the ability of Mn 2+ to support robust Tdp2 catalytic activity (20). In contrast, while co-complex structures with Ca 2+ also show a single metal ion, Ca 2+ binds in a slightly different position, shifted ∼1Å from the Mg 2+ site. Although Ca 2+ is also octahedrally coordinated, longer bond lengths for the Ca 2+ ligands (Supplementary Table S3) shift the Ca 2+ ion relative to the Mg 2+ ion site. Interestingly, bidentate inner sphere metal contacts from the Ca 2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5 -PO 4 out of the Tdp2 active site ( Figure 4D). Together with results showing that under the conditions examined here, Ca 2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca 2+ -mediated inhibition of the Tdp2 reaction.

Modeling the Tdp2 reaction coordinate
Next, to examine the feasibility of our proposed single Mg 2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog-and product-bound structures as guides. Previous structural analyses showed that the superposition of a DNA substrate mimic (5 -aminohexanol) and product (5 -PO 4 ) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5 -phosphorus (20). In this scheme ( Figure  5A), a candidate nucleophilic water that is strongly hydro- 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). A starting model was generated from atomic coordinates of the mTdp2 cat 5 -aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5aminohexanol then adding the Mg 2+ 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. After QM/MM optimization of this model ( Figure 5C, 'isubstrate'), the O-P distance is 3.4Å, which is in agreement with the range of distances observed in the mTdp2 cat 5aminohexanol substrate analog structure (3.2-3.4Å). No appreciable energy penalty is observed during the first 0.5 A of the reaction coordinate. When the reaction reaches an O-P distance of 2.18Å, formation of a transition state with an energy maximum of +7.4 kcal mol −1 is observed. Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58Å) and the phosphotyrosyl O-P distance is stretched to 1.77Å, which is 0.1Å beyond an equilibrium bond length. In the subsequent two steps of the simulation, as the water-phosphate O-P distance reduces to 1.98Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 A as the water H-O bond approaches the point of dissociation. 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. Concomitant with this, the phosphotyrosyl O-P bond weakens (d = 1.89Å), and the formation of the penta-covalent transition state ( Figure 5C 'ii-transition state') is observed. The final steps show inversion of stereochemistry at the phosphate, along with length-ening and breaking of the phosphotyrosyl O-P bond. 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.
Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation. 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 pK a of this His above 8.0 (40). 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 2+ , while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. In the final optimized structure, the observed product state ( Figure 5C, 'iiiproduct') is found in a conformation that is 7.4 kcal mol −1 more stable than the initial reactive state ( Figure 5B).
The tyrosine oxy-anion product is coordinated to the Mg 2+ ion with a 2.0Å distance, which is the shortest of the six Mg 2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg 2+ greatly stabilizes the product oxy-anion. An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. We propose this cation-interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product. Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 (30) in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing ( Figure 5D). We further examined the contribution of this cation-interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol −1 penalty in the transition state and product complex. Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol −1 penalty in the product complex. Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg 2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5 -phosphotyrosine bond hydrolysis.

Tdp2 mutational analysis
To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crys-Nucleic Acids Research, 2016, Vol. 44, No. 8 3841 tal structures (denoted with superscript numbering 'm' for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2 cat mutant proteins (denoted with superscript numbering and 'h' for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5 -Y), p-nitrophenyl phosphate (PNPP) and thymidine 5 -monophosphate pnitrophenyl ester (T5PNP) (20) (Figure 5E, Supplementary  Figures S5B and S5C). By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis. For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5 -Y and T5PNP but not PNPP that lacks a nucleobase.
Structural results and QM/MM modeling indicate m Asp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. Similar to a previously characterized h D262N mutation (20), all three substitutions ablate activity, supporting essential roles for h Asp262 ( m Asp272) in catalysis. Next, we mutated key elements of the mobile loop (␤2H␤ hydrophobic wall, Figure 2A and C). Mutations h I307A, h L305A, h L305F and h L305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). The h L305W substitution that we expect to have the most distorting impact on conformation of the ␤2H␤ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5 -Y. By comparison, as predicted by our model where ␤2H␤ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). Third, we altered properties of the proposed enzyme substrate cationinterface. No activity was detected for a mutant that removes the positive charge at this position ( h R206A). The precise geometry of this pocket is also critical for catalysis as replacement of h Arg206 ( m Arg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5 -Y, no detectable activity on T5PNP or PNPP). Similarly, mutation of h Tyr178 that structurally scaffolds the h Arg206 ( m Arg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. Fourth, we evaluated roles for the h His351-h Asp316 ( m Asp326-m His359) transition state stabilization charge pair. We found that mutations that removed the charge yet retained the ability to hydrogen bond ( h H351Q) or should abrogate the elevated pK a of the Histidine ( h D316N) had severe impacts on catalysis. Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme-substrate cation-interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.

A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function
Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons (18). We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA-protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. We identified two SNPs in human TDP2 curated in the NCBI SNP database (41) that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes h Asp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes h Ile307 for Val) ( Figure  6A). We show the h D350N substitution severely impairs activity on all substrates tested in vitro, whereas h I307V only has a mild impact on catalysis ( Figure 6B-D). To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2 cat ( Figure 6A). Interestingly, the Tdp2 single Mg 2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by h Asp350 ( m Asp358) that stabilizes the DNA-bound conformation of the ␤2H␤ substrate-binding loop through hydrogen bonding to m Trp307. Here, h Asp350 ( m Asp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.
To define the molecular basis for the h D350N ( m D358N) defect, we crystallized and determined the structure of the DNA-free form of the m D358N protein to 2.8Å resolution (PDB entry 5INN). This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. Consequently, poor electron density is visible for the ␤2H␤ loop which is mostly disordered (Supplementary Figure S6). Although Mg 2+ is present at the same concentration as the WT-mTdp cat crystals (10 mM), we find the metal site is unoccupied in the m D358N crystals. Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. In support of this, we also find that h D350N ( m D358N) impairs Mg 2+ binding as measured by intrinsic tryptophan fluorescence ( Figure 4A), and abrogates Mg 2+ -stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop ( Figure 3D).

Tdp2 facilitates NHEJ repair of 5 -phosphotyrosine adducted DSBs
Overall, our Tdp2 structure/activity studies reveal a tuned, 5 -detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway (19). Accordingly, we demonstrate here that 5tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction ( Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2 FL ( Figure 7A, lane 8). Interestingly, hTdp2 cat is slightly more effective than hTdp2 FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). We confirmed that efficient joining of the same tyrosineadducted substrate in cells ( Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). Moreover, products with error (i.e. junctions have missing sequence flanking the adducted terminus) are twice as frequent in cells deficient in Tdp2 ( Figure 7D). Therefore, in accord with previous work (19), joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).
We next compared the ability of wild-type and mutant hTdp2 FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency ( Figure 7C) and fidelity ( Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/-mouse overexpressing wild-type human Tdp2, and Tdp2 -/-MEFs overexpressing the I307V variant human Tdp2. In contrast, joining of 5 phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/-MEFs, and an equivalent defect was observed in Tdp2-/-MEFs overexpressing Tdp2 D350N. Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V ( Figure 7D). Expression of wild type or I307V human Tdp2 in Tdp2-/-MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity ( Figure 7E). The rare D350N variant is thus inactive by all metrics analyzed. By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays.

DISCUSSION
Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable (3,5,35). Understanding how cells cope with complex DNA breaks bearing topoisomerase-DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNAprotein conjugate processing: (i) The Tdp2 active site is wellsuited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg 2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing.
Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs (17), however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription (18). The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. First, the nucleic acid binding trench is assembled by a dynamic ␤2H␤ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ⑀A. This is achieved by binding of nucleic acid 'bases out' by an extended basestacking hydrophobic wall of the ␤2H␤-loop. Secondly, our QM/MM analysis further highlights an enzyme-substrate cation-interaction as an additional key feature of the Tdp2 protein-DNA crosslink binding and reversal. The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. To our knowledge, this is the first proposed example of a substrate cation-interface exploited to promote a phosphoryl-transfer reaction. This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein-RNA (42) and Hepatitis B Virus (HBV) protein-DNA processing intermediates (43). By comparison, other EEP nucleases such as Ape1 (44) and Ape2 (45) have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.
The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation. However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 ␤2H␤-loop is unknown. We hypothesize that binding of the Top2 protein component of a DNA-protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. Furthermore, highresolution structures of mouse (20) (Figures 3 and 4) and C. elegans (36) Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca 2+ in-hibition studies and QM/MM analysis that all support a feasible single Mg 2+ mediated Tdp2 catalytic mechanism.
The advent of personalized medical screening opens doors for assessment of individual vulnerabilities to commonly used chemotherapeutic drugs. It would be beneficial to employ this knowledge during the early decision making processes regarding treatment. Etoposide and other Top2 poisons remain front line anti-cancer drugs (4), and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin (18). Given Tdp2 variation in the human population, links to neurological disease (18) and viral pathogenesis (42), our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2. Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA-protein and protein-protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions.