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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.

Off-axis interferometric modules built on an ordinary bright field microscope make it possible to achieve single-shot quantitative phase imaging (QPI) by adding sufficient spatial carrier into the interferograms. However, compared with its on-axis counterparts, imaging configurations for off-axis interferometric modules have several disadvantages regarding optical aberration, stability, and space-bandwidth utilization of the lenses system. Herein, by demultiplexing technology based on polarization gratings, we propose a single-shot spatial light interference microscopy named as polarization-multiplexing light interference microscopy (PLIM) with on-axis imaging configuration to realize single-shot QPI. Although the imaging system is on-axis, the PLIM system still can generate sufficient spatial carrier, so we can adjust the orientation and absolute value of the spatial carrier independently of the magnification ratio of the imaging system. The experimental results prove that the PLIM system has better temporal phase stability compared with conventional grating-based QPI technologies and is suitable for high resolution QPI.

The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity., 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.

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.

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 theHsmar1transposase. 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 ancestralHsmar1terminal 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.

Background: Metnase, a transposase-containing DNA repair protein, retains DNA cleavage activity with a DDN motif. Results: Substitution with the ancestral transposase DDD/DDE catalytic motif results in a decrease in ssDNA binding and ss-overhang cleavage activities. Conclusion: The DDN motif is required for Metnase DNA repair activities. Significance: Understanding the requirements for catalytic activity provides insights on how Metnase functions as a DNA repair protein., Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN610 with either DDD610 or DDE610 significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN610 → DDD610, which restores the ancestral catalytic site, results in loss of function in Metnase.

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 activity 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.

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.

Secreted phospholipase A₂s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A₂ (sPLA₂-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA₂-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA₂-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA₂-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA₂-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA₂-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA₂-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA₂-IIEs.

Apurinic/apyrimidinic endonuclease I (APE1) is an essential base excision repair enzyme that catalyzes a Mg2+-dependent reaction resulting in the cleavage of the phosphodiester backbone 5′ of an abasic site within double-stranded DNA.1,2 Pre-steady-state turnover rates for APE1’s endonuclease activity have been estimated to be greater than 7003 or 850 s–1,4 whereas steady-state turnover rates are approximately 2 s–1.5 Thus, substrate turnover is diffusion-limited, and the slow step of the reaction occurs after the chemistry.4 In fact, product release has been proposed to be the slow step of the reaction.6 The endonuclease reaction involves a one-step associative phosphoryl transfer mechanism, with water serving as the nucleophile7 and preference for the Rp stereoisomer during cleavage.2 However, the number of metal ions involved and their coordination in the enzyme remains controversial. A two metal ion mechanism was first proposed by Steitz and co-workers for Klenow fragment,8,9 related polymerases, and associated exonucleases.10 Since then, there has been a general assumption that most enzymes that cleave DNA, including APE1, will in fact use two metal ions.11 APE1 is most closely related in structure to Escherichia coli exonuclease III, which binds a single metal ion in the active site.12 The two metal ion assumption was challenged by Tainer and co-workers, who put forth a mechanism for APE1 involving only one metal ion based on structural and enzymatic characterizations of an APE1–DNA complex.6 In that work, they reported a 3.0 A structure with one Mn2+ ion bound to E96 in the active site with cleaved DNA. Crystal structures of APE1 with Pb2+ bound in the active site of the enzyme were then reported, and a two metal ion mechanism was proposed.13 In this mechanism, coordinating residues for the metal ions included D70 and E96 for one metal and H309, D210, and N212 for a second metal ion, despite the fact that Mg2+ strongly prefers coordinating oxygen ligands.14 The two metal ion binding sites then formed the basis of a proposed moving metal ion mechanism involving Mg2+ binding first to a site coordinated by D210 and N212 and then moving 5 A to a site coordinated by D70, E96, and D308.15,16 Finally, a 25Mg solid-state NMR study reported that APE1 binds one mole equivalent of Mg2+, which is disordered due to its coordinating ligands, suggesting plasticity in the active site.17 Recently, a 2.4 A resolution structure of an APE1–Mg2+–product complex was reported in which Mg2+ is coordinated solely to E96,18 as was shown previously for the Mn2+ complex. Although the effect of substituting E96 on the catalytic activity was characterized in this recent study,18 there is currently no solution data for direct metal binding by APE1. In the absence of substrate, Mg2+ is coordinated by D70 and E96 in crystal structures reported to date at moderate resolution.19 In this study, we present the highest resolution structure of APE1 as a complex with bound Mg2+ determined at 1.4 A, the structure of APE1 bound to Mn2+, and the first apo-APE1 structure (i.e., without bound metal). Our motivation for this study was to establish a structural basis for metal binding in the absence of substrate and to determine the contributions of specific residues on metal binding and catalysis by APE1. Our results provide new insights on the initial capture of metal ion by APE1 involving remarkable plasticity of a metal-coordinating ligand within the active site.

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 IC(50) 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.

Secreted phospholipase A₂s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A₂ (sPLA₂-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA₂-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA₂-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA₂-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA₂-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA₂-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA₂-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA₂-IIEs.

SETMAR, a recently identified double strand break (DSB) repair enzyme in the human genome, contains an N-terminal SET domain and a C-terminal MAR domain. This chimeric protein arose through the fusion of a mariner-family DNA transposase gene, Hsmar1, downstream of a SET histone methyltransferase gene approximately 50 million years ago [1]. Although the SETMAR transposase domain retains the ability to bind terminal inverted repeat (TIR) DNA sequence, which is the hallmark of DNA transposons, it is no longer a functional transposase [2]. Nonetheless, the transposase domain with only 19 amino acid substitutions as compared to the ancestral Hsmar1 transposase has been under a strong selective evolutionary pressure suggesting that the transposase domain is functionally important. Determining how SETMAR interacts with DNA is central to understanding the molecular basis of its evolved DNA repair activity. Toward this goal, we have focused initially on the interaction of the DNA-binding domain (DBD) of the SETMAR transposase domain with TIR DNA. The DBD of SETMAR has been overexpressed and purified. A complex formed between SETMAR DBD and its transposon TIR DNA has been crystallized by using the hanging-drop vapor diffusion method. The crystals diffract to 3.15 Å resolution and exhibit orthorhombic symmetry (C2221), with unit-cell dimensions of a=72.233 Å, b=164.385 Å, and c=67.957 Å. As there is no suitable search model available, we are currently pursuing experimental phasing approaches in order to solve this structure. We anticipate the structural analysis of DBD of SETMAR bound to transposon DNA will provide insight into the mechanism by which SETMAR recognizes both TIR and non-TIR DNA.