Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA.

The binding interaction between the DNA repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) with promoter G-quadruplex (G4) folds bearing an abasic site (AP) can serve as a gene regulatory switch during oxidative stress. Prior fluorescence-based analysis in solution suggested APE1 binds the VEGF promoter G4 but whether this interaction was specific or not remained an open question. Second harmonic generation (SHG) was used in this work to measure the noncanonical DNA-protein binding interaction in a label-free assay with high sensitivity to demonstrate the interaction is ordered and specific. The binding of APE1 to the VEGF promoter G4 with AP sites modeled by a tetrahydrofuran analogue produced dissociation constants of ∼100 nM that differed from duplex and single-stranded DNA control studies. The SHG measurements confirmed APE1 binds the VEGF G4 folds in a specific manner resolving a remaining question regarding how this endonuclease with gene regulatory features engages G4 folds. The studies demonstrate the power of SHG to interrogate noncanonical DNA-protein interactions providing a foundational example for the use of this analytical method in future biochemical analyses.

The Fifth Domain in the G-Quadruplex-Forming Sequence of the Human NEIL3 Promoter Locks DNA Folding in Response to Oxidative Damage.

DNA oxidation is an inevitable and usually detrimental process, but the cell is capable of reversing this state because the cell possesses a highly developed set of DNA repair machineries, including the DNA glycosylase NEIL3 that is encoded by the NEIL3 gene. In this work, the G-rich promoter region of the human NEIL3 gene was shown to fold into a dynamic G-quadruplex (G4) structure under nearly physiological conditions using spectroscopic techniques (e.g., nuclear magnetic resonance, circular dichroism, fluorescence, and ultraviolet-visible) and DNA polymerase stop assays. The presence of 8-oxo-7,8-dihydroguanine (OG) modified the properties of the NEIL3 G4 and entailed the recruitment of the fifth domain to function as a "spare tire", in which an undamaged fifth G-track is swapped for the damaged section of the G4. The polymerase stop assay findings also revealed that owing to its dynamic polymorphism, the NEIL3 G4 is more readily bypassed by DNA polymerase I (Klenow fragment) than well-known oncogene G4s are. This study identifies the NEIL3 promoter possessing a G-rich element that can adopt a G4 fold, and when OG is incorporated, the sequence can lock into a more stable G4 fold via recruitment of the fifth track of Gs.

Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom.

The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C1-C4, C2-C6, C3-C5. The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC50, 10.2 µM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.

NEIL3 promoter G-quadruplex with oxidatively modified bases shows magnesium-dependent folding that stalls polymerase bypass.

Oxidative stress unleashes reactive species capable of oxidizing 2'-deoxyguanosine (G) nucleotides in G-rich sequences of the genome, such as the potential G-quadruplex forming sequencing (PQS) in the NEIL3 gene promoter. Oxidative modification of G yields 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) that can be further oxidized to hydantoin products. Herein, OG was synthesized into the NEIL3 PQS that was allowed to fold to a G-quadruplex (G4) in K+ ion solutions with varying amounts of Mg2+ in the physiological range. The Mg2+ dependency in the oxidatively modified NEIL3 G4 to stall a replicative DNA polymerase was evaluated. The polymerase was found to stall at the G4 or OG, as well as continue to full-length extension with dependency on the location of the modification and the concentration of Mg2+. To provide some clarity on these findings, OG or the hydantoins were synthesized in model NEIL3 G4 folding sequences at the positions of the polymerase study. The model G4 sequences were allowed to fold in K+ ion solutions with varying levels of Mg2+ to identify how the presence of the divalent metal impacted G4 folding depending on the location of the modification. The presence of Mg2+ either caused the transition of the NEIL3 G4 folds from an antiparallel to parallel orientation of the strands or had no impact. Structural models are proposed to understand the findings using the literature as a guide. The biological significance of the results is discussed.

Synechococcus elongatus Argonaute reduces natural transformation efficiency and provides immunity against exogenous plasmids.

IMPORTANCE: S. elongatus is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used for genetic engineering to produce renewable biochemicals. Our findings reveal an SeAgo-based defense mechanism in S. elongatus against the horizontal transfer of genetic material. We demonstrate that deletion of the ago gene facilitates genetic studies and genetic engineering of S. elongatus.

Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from Clavus davidgilmouri (Gastropoda: Conoidea: Drilliidae).

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Clavus davidgilmouri Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.