Mardiyyah/TAPT-PDBE-V1 · Datasets at Hugging Face

PMCID stringclasses 30 values	Title stringclasses 30 values	Sentences stringlengths 2 1.94k
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	We propose this cation–π interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	We further examined the contribution of this cation–π 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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol penalty in the transition state and product complex.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol penalty in the product complex.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2 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 p-nitrophenyl ester (T5PNP) (20) (Figure 5E, Supplementary Figures S5B and S5C).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Structural results and QM/MM modeling indicate Asp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Similar to a previously characterized D262N mutation (20), all three substitutions ablate activity, supporting essential roles for Asp262 (Asp272) in catalysis.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Mutations I307A, L305A, L305F and L305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	The L305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Third, we altered properties of the proposed enzyme substrate cation–π interface.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	No activity was detected for a mutant that removes the positive charge at this position (R206A).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	The precise geometry of this pocket is also critical for catalysis as replacement of Arg206 (Arg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Similarly, mutation of Tyr178 that structurally scaffolds the Arg206 (Arg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Fourth, we evaluated roles for the His351–Asp316 (Asp326–His359) transition state stabilization charge pair.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	We found that mutations that removed the charge yet retained the ability to hydrogen bond (H351Q) or should abrogate the elevated pKa of the Histidine (D316N) had severe impacts on catalysis.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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 Asp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes Ile307 for Val) (Figure 6A).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	We show the D350N substitution severely impairs activity on all substrates tested in vitro, whereas I307V only has a mild impact on catalysis (Figure 6B–D).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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 (Figure 6A).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Interestingly, the Tdp2 single Mg ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by Asp350 (Asp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to Trp307.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Here, Asp350 (Asp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Tdp2 SNPs impair function. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	A) Active site residues mutated by TDP2 SNPs.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2 structure (4GZ1). (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2 proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2 proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	D) Relative activity of WT and indicated mutant human MBP-hTdp2 fusion proteins on three model Tdp2 substrates.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Quantification of percent MBP-hTdp2 activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Error bars, s.d.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	n = 3.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	To define the molecular basis for the D350N (D358N) defect, we crystallized and determined the structure of the DNA-free form of the D358N protein to 2.8Å resolution (PDB entry 5INN).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Although Mg is present at the same concentration as the WT-mTdp crystals (10 mM), we find the metal site is unoccupied in the D358N crystals.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	In support of this, we also find that D350N (D358N) impairs Mg binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Accordingly, we demonstrate here that 5′-tyrosylated 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 (Figure 7A, lane 8).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Interestingly, hTdp2 is slightly more effective than hTdp2 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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	We confirmed that efficient joining of the same tyrosine-adducted 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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2 as indicated (+) for 5 min at 37°C.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Concatemer ligation products were detected by 5% native PAGE. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	B) Workflow diagram of cellular end joining assays.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2 cells and Tdp2 cells complemented with wildtype or the noted hTDP2 variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Error bars, s.d, n = 3. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Error bars, s.d, n = 3. (
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	We next compared the ability of wild-type and mutant hTdp2 variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by 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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	The rare D350N variant is thus inactive by all metrics analyzed.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited 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 cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	To our knowledge, this is the first proposed example of a substrate cation–π interface exploited to promote a phosphoryl-transfer reaction.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Furthermore, high-resolution 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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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 inhibition studies and QM/MM analysis that all support a feasible single Mg mediated Tdp2 catalytic mechanism.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	The advent of personalized medical screening opens doors for assessment of individual vulnerabilities to commonly used chemotherapeutic drugs.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	It would be beneficial to employ this knowledge during the early decision making processes regarding treatment.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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).
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by 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.
PMC4857006	Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.	Coordinates and structure factors have been deposited in the RCSB Protein Data Bank under accession code 5HT2 (mTdp2-Mg-ϵA-DNA complex), 5INK (mTdp2-Mg-THF complex), 5INL (mTdp2-Mg-dA-DNA-product complex), 5INM (mTdp2-apo structure), 5INN (mTdp2-D350N structure), 5INO (hTdp2-Mg-DNA product complex), 5INP (mTdp2-Mn-DNA product complex) and 5INQ (mTdp2-Ca-DNA product complex).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

We propose this cation–π interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

We further examined the contribution of this cation–π 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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol penalty in the transition state and product complex.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol penalty in the product complex.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2 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 p-nitrophenyl ester (T5PNP) (20) (Figure 5E, Supplementary Figures S5B and S5C).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Structural results and QM/MM modeling indicate Asp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Similar to a previously characterized D262N mutation (20), all three substitutions ablate activity, supporting essential roles for Asp262 (Asp272) in catalysis.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Mutations I307A, L305A, L305F and L305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

The L305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Third, we altered properties of the proposed enzyme substrate cation–π interface.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

No activity was detected for a mutant that removes the positive charge at this position (R206A).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

The precise geometry of this pocket is also critical for catalysis as replacement of Arg206 (Arg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Similarly, mutation of Tyr178 that structurally scaffolds the Arg206 (Arg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Fourth, we evaluated roles for the His351–Asp316 (Asp326–His359) transition state stabilization charge pair.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

We found that mutations that removed the charge yet retained the ability to hydrogen bond (H351Q) or should abrogate the elevated pKa of the Histidine (D316N) had severe impacts on catalysis.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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 Asp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes Ile307 for Val) (Figure 6A).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

We show the D350N substitution severely impairs activity on all substrates tested in vitro, whereas I307V only has a mild impact on catalysis (Figure 6B–D).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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 (Figure 6A).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Interestingly, the Tdp2 single Mg ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by Asp350 (Asp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to Trp307.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Here, Asp350 (Asp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Tdp2 SNPs impair function. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

A) Active site residues mutated by TDP2 SNPs.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2 structure (4GZ1). (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2 proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2 proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

D) Relative activity of WT and indicated mutant human MBP-hTdp2 fusion proteins on three model Tdp2 substrates.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Quantification of percent MBP-hTdp2 activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Error bars, s.d.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

n = 3.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

To define the molecular basis for the D350N (D358N) defect, we crystallized and determined the structure of the DNA-free form of the D358N protein to 2.8Å resolution (PDB entry 5INN).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Although Mg is present at the same concentration as the WT-mTdp crystals (10 mM), we find the metal site is unoccupied in the D358N crystals.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

In support of this, we also find that D350N (D358N) impairs Mg binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Accordingly, we demonstrate here that 5′-tyrosylated 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 (Figure 7A, lane 8).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Interestingly, hTdp2 is slightly more effective than hTdp2 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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

We confirmed that efficient joining of the same tyrosine-adducted 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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2 as indicated (+) for 5 min at 37°C.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Concatemer ligation products were detected by 5% native PAGE. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

B) Workflow diagram of cellular end joining assays.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2 cells and Tdp2 cells complemented with wildtype or the noted hTDP2 variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Error bars, s.d, n = 3. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Error bars, s.d, n = 3. (

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

We next compared the ability of wild-type and mutant hTdp2 variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by 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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

The rare D350N variant is thus inactive by all metrics analyzed.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited 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 cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

To our knowledge, this is the first proposed example of a substrate cation–π interface exploited to promote a phosphoryl-transfer reaction.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Furthermore, high-resolution 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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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 inhibition studies and QM/MM analysis that all support a feasible single Mg mediated Tdp2 catalytic mechanism.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

The advent of personalized medical screening opens doors for assessment of individual vulnerabilities to commonly used chemotherapeutic drugs.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

It would be beneficial to employ this knowledge during the early decision making processes regarding treatment.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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).

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by 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.

PMC4857006

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Coordinates and structure factors have been deposited in the RCSB Protein Data Bank under accession code 5HT2 (mTdp2-Mg-ϵA-DNA complex), 5INK (mTdp2-Mg-THF complex), 5INL (mTdp2-Mg-dA-DNA-product complex), 5INM (mTdp2-apo structure), 5INN (mTdp2-D350N structure), 5INO (hTdp2-Mg-DNA product complex), 5INP (mTdp2-Mn-DNA product complex) and 5INQ (mTdp2-Ca-DNA product complex).