Etienne Henry

A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain

HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3′-processing reaction specificity—the first reaction of the integration process—at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase–DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg2+ cofactor. The DNA-binding was essentially non-cooperative with Mn2+ or using non-specific/random sequences, regardless of the metallic cofactor. 2,2′-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg2+-mediated integrase–DNA interaction, without affecting the overall affinity. Concomitantly, 2,2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC50 = 11–15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2′-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn2+-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg2+ cofactor and the zinc effector.

Mitochondria-targeted Triphenylamine Derivatives Activatable by Two-Photon Excitation for Triggering and Imaging Cell Apoptosis

Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760–860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.

Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: a fluorescence cross-correlation spectroscopy study.

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3′-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 °C, whereas only modest or no cooperativity was observed at 25 °C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 °C but not at 25 °C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.

Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation.

We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push–pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin–autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800–950-nm range, with great perspectives for eNOS imaging in living tissues.

Synthesis and spectral properties of new fluorescent alkoxy-substituted thieno[3,2-b]indole derivatives ☆

The synthesis and optical properties of three new fluorescent alkoxy-substituted thieno[3,2-b]indole (TI) derivatives, including 7-methoxy thieno[3,2-b]indole (7-MeOTI), 6,7- methylenedioxythieno[3,2-b]indole (6,7-MDTI) and 6,7-dihexyloxythieno[3,2-b]indole, (6,7-DHTI), were investigated. Electronic absorption spectra, fluorescence excitation and emission spectra, fluorescence quantum yields (ΦF), lifetimes (τF), and other photophysical parameters of the three TI derivatives were measured in DMSO solutions at room temperature. Theoretical electronic absorption and fluorescence spectra were also calculated by means of a molecular orbital (MO) method. For all three alkoxy-TI derivatives, the fluorescence emission maximum wavelength was significantly red shifted relative to un-substituted TI, which was attributed to delocalization of the fused hetero-aromatic ring π electronic system by the electron-donating alkoxy group(s). ΦF values varied from 0.12 to 0.19, according to the compound. τF were short, in the range 0.56-1.13 ns.

Mechanistic insight into cadmium-induced inactivation of the Bloom protein.

Cadmium is a toxic metal that inactivates DNA-repair proteins via multiple mechanisms, including zinc substitution. In this study, we investigated the effect of Cd2+ on the Bloom protein (BLM), a DNA-repair helicase carrying a zinc-binding domain (ZBD) and playing a critical role to ensure genomic stability. One characteristics of BLM-deficient cells is the elevated rate of sister chromatid exchanges, a phenomenon that is also induced by Cd2+. Here, we show that Cd2+ strongly inhibits both ATPase and helicase activities of BLM. Cd2+ primarily prevents BLM-DNA interaction via its binding to sulfhydryl groups of solvent-exposed cysteine residues and, concomitantly, promotes the formation of large BLM multimers/aggregates. In contrast to previously described Cd2+ effects on other zinc-containing DNA-repair proteins, the ZBD appears to play a minor role in the Cd2+-mediated inhibition. While the Cd2+-dependent formation of inactive multimers and the defect of DNA-binding were fully reversible upon addition of EDTA, the inhibition of the DNA unwinding activity was not counteracted by EDTA, indicating another mechanism of inhibition by Cd2+ relative to the targeting of a catalytic residue. Altogether, our results provide new clues for understanding the mechanism behind the ZBD-independent inactivation of BLM by Cd2+ leading to accumulation of DNA double-strand breaks.

An experimental study of the electronic absorption and fluorescence spectral properties of new p-substituted-N-phenylpyrroles and their electrosynthesized polymers

Electronic absorption and fluorescence spectral properties of new p-substituted-N-phenylpyrroles (N-PhPys), including HOPhPy, MeOPhPy, ThPhPy, PhDPy, DPhDPy, PyPhThThPhPy, and their available, electrosynthesized polymers were investigated. Electronic absorption spectra, fluorescence excitation and emission spectra, fluorescence quantum yields (ΦF) and lifetimes (τF), and other photophysical parameters of these N-PhPy derivatives and their polymers were measured in DMF, DMSO diluted solutions and/or solid state at room temperature. The electronic absorption spectra of N-PhPy derivatives and their polymers included one to several bands, located in the 270-395 nm region, according to the p-phenyl substituent electron-donating effect and conjugated heteroaromatic system length. The fluorescence excitation spectra were characterized by one broad main peak, with, in most cases, one (or more) poorly resolved shoulder (s), appearing in the 270-405 nm region, and their emission spectra were generally constituted of several bands located in the 330-480 nm region. No significant shift of the absorption, fluorescence excitation and emission spectra wavelengths was found upon going from the monomers to the corresponding polymers. ΦF values were high, varying between 0.11 and 0.63, according to the nature of substituents(s) and to the conjugated system extension. Fluorescence decays were mono-exponential for the monomers and poly-exponential for PyPhThThPhPy and for polymers. τF values were relatively short (0.35-5.17 ns), and markedly decreased with the electron-donor character of the phenyl group p-substituent and the conjugated system extension.

Physical and functional interplay between PCNA DNA clamp and Mre11–Rad50 complex from the archaeon Pyrococcus furiosus

Abstract Several archaeal species prevalent in extreme environments are particularly exposed to factors likely to cause DNA damages. These include hyperthermophilic archaea (HA), living at temperatures >70°C, which arguably have efficient strategies and robust genome guardians to repair DNA damage threatening their genome integrity. In contrast to Eukarya and other archaea, homologous recombination appears to be a vital pathway in HA, and the Mre11–Rad50 complex exerts a broad influence on the initiation of this DNA damage response process. In a previous study, we identified a physical association between the Proliferating Cell Nuclear Antigen (PCNA) and the Mre11–Rad50 (MR) complex. Here, by performing co-immunoprecipitation and SPR analyses, we identified a short motif in the C- terminal portion of Pyrococcus furiosus Mre11 involved in the interaction with PCNA. Through this work, we revealed a PCNA-interaction motif corresponding to a variation on the PIP motif theme which is conserved among Mre11 sequences of Thermococcale species. Additionally, we demonstrated functional interplay in vitro between P. furiosus PCNA and MR enzymatic functions in the DNA end resection process. At physiological ionic strength, PCNA stimulates MR nuclease activities for DNA end resection and promotes an endonucleolytic incision proximal to the 5′ strand of double strand DNA break.

Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

Abstract Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequences of replacing Mg2+ by Ca2+ on base pairing at the polymerase active site as well as the editing of terminal nucleotides at the exonuclease active site of the archaeal Pyrococcus abyssi DNA Pol (PabPolB) are characterized and compared to other (families B, A, Y, X, D) DNA Pols. Based on primer extension assays, steady-state kinetics and ion-chased experiments, we demonstrate that Ca2+ (and other metal ions) activates DNA synthesis by PabPolB. While showing a slower rate of phosphodiester bond formation, nucleotide selectivity is improved over that of Mg2+. Further mechanistic studies show that the affinities for primer/template are higher in the presence of Ca2+ and reinforced by a correct incoming nucleotide. Conversely, no exonuclease degradation of the terminal nucleotides occurs with Ca2+. Evolutionary and mechanistic insights among DNA Pols are thus discussed.