Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

The DNA-binding domain of SETMAR was successfully crystallized in a complex with its ancestral terminal inverted repeat and a variant of this sequence through a systematic approach, and initial Se SAD phasing was achieved through the judicious addition of Met residues., Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts

A role for SETMAR in gene regulation: insights from structural analysis of the dna-binding domain in complex with dna

Indiana University-Purdue University Indianapolis (IUPUI)SETMAR is a chimeric protein that originates from the fusion of a SET domain to the mariner Hsmar1 transposase. This fusion event occurred approximately 50 million years ago, after the split of an anthropoid primate ancestor from the prosimians. Thus, SETMAR is only expressed in anthropoid primates, such as humans, apes, and New World monkeys. Evolutionary sequence analyses have revealed that the DNA-binding domain, one of the two functional domains in the Hsmar1 transposase, has been subjected to a strong purifying selection. Consistent with these analyses, SETMAR retains robust binding specificity to its ancestral terminal inverted repeat (TIR) DNA. In the human genome, this TIR sequence is dispersed in over 1500 perfect or nearly perfect sites. Given that many DNA-binding domains of transcriptional regulators are derived from transposases, we hypothesized that SETMAR may play a role in gene regulation. In this thesis, we determined the crystal structures of the DNA-binding domain bound to both its ancestral TIR DNA and a variant TIR DNA sequence at 2.37 and 3.07 Å, respectively. Overall, the DNA-binding domain contains two helix-turn-helix (HTH) motifs linked by two AT-hook motifs and dimerizes through its HTH1 motif. In both complexes, minor groove interactions with the AT-hook motifs are similar, and major groove interactions with HTH1 involve a single residue. However, four residues from HTH2 participate in nucleobase-specific interactions with the TIR and only two with the variant DNA sequence. Despite these differences in nucleobase-specific interactions, the DNA-binding affinities of SETMAR to TIR or variant TIR differ by less than two-fold. From cell-based studies, we found that SETMAR represses firefly luciferase gene expression while the DNA-binding deficient mutant does not. A chromatin immunoprecipitation assay further confirms that SETMAR binds the TIR sequence in cells. Collectively, our studies suggest that SETMAR functions in gene regulation

High-Resolution Crystal Structures Reveal Plasticity in the Metal Binding Site of Apurinic/Apyrimidinic Endonuclease I

Apurinic/apyrimidinic endonuclease I (APE1) is an essential base excision repair enzyme that catalyzes a Mg2+-dependent reaction in which the phosphodiester backbone is cleaved 5′ of an abasic site in duplex DNA. This reaction has been proposed to involve either one or two metal ions bound to the active site. In the present study, we report crystal structures of Mg2+, Mn2+, and apo-APE1 determined at 1.4, 2.2, and 1.65 Å, respectively, representing two of the highest resolution structures yet reported for APE1. In our structures, a single well-ordered Mn2+ ion was observed coordinated by D70 and E96; the Mg2+ site exhibited disorder modeled as two closely positioned sites coordinated by D70 and E96 or E96 alone. Direct metal binding analysis of wild-type, D70A, and E96A APE1, as assessed by differential scanning fluorimetry, indicated a role for D70 and E96 in binding of Mg2+ or Mn2+ to APE1. Consistent with the disorder exhibited by Mg2+ bound to the active site, two different conformations of E96 were observed coordinated to Mg2+. A third conformation for E96 in the apo structure is similar to that observed in the APE1–DNA–Mg2+ complex structure. Thus, binding of Mg2+ in three different positions within the active site of APE1 in these crystal structures corresponds directly with three different conformations of E96. Taken together, our results are consistent with the initial capture of metal by D70 and E96 and repositioning of Mg2+ facilitated by the structural plasticity of E96 in the active site

Discovery of macrocyclic inhibitors of apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential base excision repair enzyme that is upregulated in a number of cancers, contributes to resistance of tumors treated with DNA-alkylating or -oxidizing agents, and has recently been identified as an important therapeutic target. In this work, we identified hot spots for binding of small organic molecules experimentally in high resolution crystal structures of APE1 and computationally through the use of FTMAP analysis (http://ftmap.bu.edu/). Guided by these hot spots, a library of drug-like macrocycles was docked and then screened for inhibition of APE1 endonuclease activity. In an iterative process, hot-spot-guided docking, characterization of inhibition of APE1 endonuclease, and cytotoxicity of cancer cells were used to design next generation macrocycles. To assess target selectivity in cells, selected macrocycles were analyzed for modulation of DNA damage. Taken together, our studies suggest that macrocycles represent a promising class of compounds for inhibition of APE1 in cancer cells.This work was supported by grants from the National Institutes of Health (Grant R01CA205166 to M.R.K. and M.M.G. and Grant R01CA167291 to M.R.K.) and by the Earl and Betty Herr Professor in Pediatric Oncology Research, Jeff Gordon Children's Foundation, and the Riley Children's Foundation (M.R.K.). Work at the BU-CMD (J.A.P., L.E.B., R.T.) is supported by the National Institutes of Health, Grant R24 GM111625. D.B. and S.V. were supported by the National Institutes of Health, Grant R35 GM118078. (R35 GM118078 - National Institutes of Health; R01CA205166 - National Institutes of Health; R01CA167291 - National Institutes of Health; R24 GM111625 - National Institutes of Health; Earl and Betty Herr Professor in Pediatric Oncology Research; Jeff Gordon Children's Foundation; Riley Children's Foundation)Accepted manuscriptSupporting documentatio

Characterization of the redox activity and disulfide bond formation in Apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1’s redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1’s redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1–TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors

Small molecule activation of apurinic/apyrimidinic endonuclease 1 reduces DNA damage induced by cisplatin in cultured sensory neurons

Although chemotherapy-induced peripheral neuropathy (CIPN) affects approximately 5-60% of cancer patients, there are currently no treatments available in part due to the fact that the underlying causes of CIPN are not well understood. One contributing factor in CIPN may be persistence of DNA lesions resulting from treatment with platinum-based agents such as cisplatin. In support of this hypothesis, overexpression of the base excision repair (BER) enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), reduces DNA damage and protects cultured sensory neurons treated with cisplatin. Here, we address stimulation of APE1's endonuclease through a small molecule, nicorandil, as a means of mimicking the beneficial effects observed for overexpression of APE1. Nicorandil, was identified through high-throughput screening of small molecule libraries and found to stimulate APE1 endonuclease activity by increasing catalytic efficiency approximately 2-fold. This stimulation is primarily due to an increase in kcat. To prevent metabolism of nicorandil, an approved drug in Europe for the treatment of angina, cultured sensory neurons were pretreated with nicorandil and daidzin, an aldehyde dehydrogenase 2 inhibitor, resulting in decreased DNA damage but not altered transmitter release by cisplatin. This finding suggests that activation of APE1 by nicorandil in cisplatin-treated cultured sensory neurons does not imbalance the BER pathway in contrast to overexpression of the kinetically faster R177A APE1. Taken together, our results suggest that APE1 activators can be used to reduce DNA damage induced by cisplatin in cultured sensory neurons, although further studies will be required to fully assess their protective effects

Inhibition of Apurinic/apyrimidinic endonuclease I’s redox activity revisited

The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen–deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 μM are required to inhibit APE1’s endonuclease activity. To determine the role of E3330’s negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1’s redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC50 values (8.5 and 7 μM) lower than those produced with E3330 (20 and 55 μM, respectively). Thus, E3330’s negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1

DELAY OF GERMINATION 1, the Master Regulator of Seed Dormancy, Integrates the Regulatory Network of Phytohormones at the Transcriptional Level to Control Seed Dormancy

Seed dormancy, an important adaptive trait that governs germination timing, is endogenously controlled by phytohormones and genetic factors. DELAY OF GERMINATION 1 (DOG1) is the vital genetic regulator of dormancy, significantly affecting the expression of numerous ABA and GA metabolic genes. However, whether DOG1 could influence the expression of other phytohormone-related genes is still unknown. Here, we comprehensively investigated all well-documented hormone-related genes which might be affected in dog1–2 dry or imbibed seeds by using whole-transcriptome sequencing (RNA-seq). We found that DOG1 could systematically control the expression of phytohormone-related genes. An evident decrease was observed in the endogenous signal intensity of abscisic acid (ABA) and indole-3-acetic acid (IAA), while a dramatic increase appeared in that of gibberellins (GA), brassinosteroids (BR), and cytokinin (CK) in the dog1–2 background, which may contribute considerably to its dormancy-deficient phenotype. Collectively, our data highlight the role of DOG1 in balancing the expression of phytohormone-related genes and provide inspirational evidence that DOG1 may integrate the phytohormones network to control seed dormancy

Structural basis of seamless excision and specific targeting by piggyBac transposase

PiggyBac is a transposon used in genome engineering that does not leave excision footprints. Here the authors determine the structures of two complexes in which the piggyBac transposase is bound to DNA representing different steps of the transposition reaction, providing a basis for how the transposition reaction proceeds

Structural and genome-wide analyses suggest that transposon-derived protein SETMAR alters transcription and splicing

Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (∼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre-mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development