Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.     

Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.     

Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1

The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl–DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD⁺. The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1–PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.     

New inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties

A number of derivatives of 7-hydroxycoumarins containing aromatic or monoterpene substituents at hydroxy-group were synthesized based on a hit compound from a virtual screen. The ability of these compounds to inhibit tyrosyl-DNA phosphodiesterase I (Tdp 1), important target for anti-cancer therapy, was studied for the first time. It was found that the 7-hydroxycoumarin derivatives with monoterpene pinene moiety are effective inhibitors of Tdp 1 with the most active derivative (+)-25c with IC₅₀ value of 0.675 μM. This compound has low cytotoxicity (CC₅₀ >100 μM) when tested against human cancer cells which is crucial for presupposed application in combination with clinically established anticancer drugs. The ability of the new compounds to enhance the cytotoxicity of camptothecin, an established topoisomerase 1 poison, was demonstrated.     

Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3'-phosphate. The only known example of such a linkage in eukaryotic cells occurs normally as a transient link between a type IB topoisomerase and DNA. Thus human Tdp1 is thought to be responsible for repairing lesions that occur when topoisomerase I becomes stalled on the DNA in the cell. Tdp1 has also been shown to remove glycolate from single-stranded DNA containing a 3'-phosphoglycolate, suggesting a role for Tdp1 in repair of free-radical mediated DNA double-strand breaks. We report the three-dimensional structures of human Tdp1 bound to the phosphate transition state analogs vanadate and tungstate. Each structure shows the inhibitor covalently bound to His263, confirming that this residue is the nucleophile in the first step of the catalytic reaction. Vanadate in the Tdp1-vanadate structure has a trigonal bipyramidal geometry that mimics the transition state for hydrolysis of a phosphodiester bond, while Tdp1-tungstate displays unusual octahedral coordination. The presence of low-occupancy tungstate molecules along the narrow groove of the substrate binding cleft is suggestive evidence that this groove binds ssDNA. In both cases, glycerol from the cryoprotectant solution became liganded to the vanadate or tungstate inhibitor molecules in a bidentate 1,2-diol fashion. These structural models allow predictions to be made regarding the specific binding mode of the substrate and the mechanism of catalysis.     

Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1

TDP (tyrosyl-DNA phosphodiesterase) 1 catalyses the hydrolysis of phosphodiester linkages between a DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel-shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nt indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has been found previously that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid topoisomerase I-derived peptide were efficiently cleaved by TDP1, but similar to the full-length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo.     

TDP1 is required for efficient non-homologous end joining in human cells

Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3′ and 5′ terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-SceI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81.     

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.     

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.     

Purification and properties of two endonucleases specific for apurinic/apyrimidinic sites in DNA from Saccharomyces cerevisiae

Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24 000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca²⁺ or Mg²⁺ and AP-endonuclease B by Mg²⁺ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonuclease B at pH 8. AP-endonuclease A is more stable at 60°C (half-life of 17 min) than B (half-life of 4 min). AP-endonucleuase A is insensitive to N-ethylmaleimide or ρ-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but ρ-chloromercuribenzoate inhibits its activity.     

Apurinic/apyrimidinic endonuclease 1, the sensitive marker for DNA deterioration in dextran sulfate sodium-induced acute colitis

Abstract

Mutations in mismatch repair (MMR) genes are commonly associated with the development of colorectal cancer. Additionally, base excision repair, which involves apurinic/apyrimidinic endonuclease 1 (APE1), recognizes and eliminates oxidative DNA damage. Here, we investigated the possible roles of APE1 in dextran sulfate sodium (DSS)-induced acute colitis using the young rat model. Four-week-old Sprague–Dawley rats were administered 2% DSS in drinking water for 1 week. MMR and APE1 expression levels were assessed by western blotting and immunohistochemistry. Following DSS treatment, growth of young rats failed and the animals had loose stools. Together with the histological changes associated with acute colitis, APE1 and MSH2 levels increased significantly at 3 and 5 days after DSS treatment, respectively. The difference between APE1 and MSH2 expression was significant. DSS-induced DNA damage and subsequent repair activity were evaluated by staining for 8-hydroxy-deoxyguanosine (8-OHdG) and APE1, respectively; 8-OHdG immunoreactivity increased throughout the colonic mucosa, while APE1 levels in the surface epithelium increased at an earlier timepoint. Taken together, our data suggest that changes in APE1 expression after DSS treatment occurred earlier and were more widespread than changes in MMR expression, suggesting that APE1 is more sensitive for prediction of DNA deterioration in DSS-induced colitis.     

The Trypanosoma cruzi L1Tc and NARTc non-LTR retrotransposons show relative site specificity for insertion

The trypanosomatid protozoan Trypanosoma cruzi contains long autonomous (L1Tc) and short nonautonomous (NARTc) non-long terminal repeat retrotransposons. NARTc (0.25 kb) probably derived from L1Tc (4.9 kb) by 3'-deletion. It has been proposed that their apparent random distribution in the genome is related to the L1Tc-encoded apurinic/apyrimidinic endonuclease (APE) activity, which repairs modified residues. To address this question we used the T. cruzi (CL-Brener strain) genome data to analyze the distribution of all the L1Tc/NARTc elements present in contigs larger than 10 kb. This data set, which represents 0.91x sequence coverage of the haploid nuclear genome ( approximately 55 Mb), contains 419 elements, including 112 full-length L1Tc elements (14 of which are potentially functional) and 84 full-length NARTc. Approximately half of the full-length elements are flanked by a target site duplication, most of them (87%) are 12 bp long. Statistical analyses of sequences flanking the full-length elements show the same highly conserved pattern upstream of both the L1Tc and NARTc retrotransposons. The two most conserved residues are a guanine and an adenine, which flank the site where first-strand cleavage is performed by the element-encoded endonuclease activity. This analysis clearly indicates that the L1Tc and NARTc elements display relative site specificity for insertion, which suggests that the APE activity is not responsible for first-strand cleavage of the target site.     

Some properties of the interstrand crosslinks in depurinated DNA

The interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.     

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5′-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5′ direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3′ direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3′ direction (AP₊₃-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP₊₃-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3′ → 5′ exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.     

Monoclonal antibodies targeting the synthetic peptide corresponding to the polybasic cleavage site on H5N1 influenza hemagglutinin

Background

Avian influenza H5N1 virus is highly pathogenic partially because its H5 hemagglutinin contains a polybasic cleavage site that can be processed by proteases in multiple organs.

Methods

Monoclonal antibodies (mAb) specific to the synthetic peptide of hemagglutinin polybasic cleavage site of H5N1 virus were raised and tested for their neutralizing potential.

Results

Purified mAb showed suppression of H5N1 pseudovirus infection on Madin-Darby Canine Kidney (MDCK) cells but the efficacy was less than 50%. Since those mAb are specific to the intact uncut polybasic cleavage site of hemagglutinin, their efficacy depends on the extent of hemagglutinin cleavage on the viral surface.

Conclusions

Proteolytic analysis suggests the low efficacy associated with those mAb may be due to proteolytic cleavage already present on the majority of hemagglutinin prior to the infection of virus.     

The cleavage initiation site establishes the posterior pole of the hydrozoan embryo

Summary When the first cleavage of the hydrozoan egg is reversibly suppressed, two cleavage furrows frequently form simultaneously at the time of the second cleavage. If these two cleavage initiation sites are far enough apart, each one specifies a site of gastrulation, and the embryo that forms develops into a two tailed planula larva. When two tailed planulae are induced to metamorphose, they form a polyp with two stalks and hydranths.     

The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: Stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue

Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding.