NMR evidence for specific intercalation of Δ-rh(phen)_2phi^(3+) in [d(GTCGAC)_2]

The anchoring of metal complexes in the major groove of DNA through intercalation has been increasingly useful in the shape-selective design of novel metal complexes which bind DNA with high sequence-selectivity. Toward that goal, direct structural information regarding this intercalative interaction is essential. Phenanthrenequinone diimine (phi) complexes of Rh(III) bind avidly (K_b 1 ≥ 10^7) to DNA by intercalation in the major groove. Here we report the first ^1H-NMR studies of Δ-rh(phen)_2phi^(3+) bound to an oligonucleotide. These studies provide direct structural evidence for specific intercalation by this octahedral complex in the major groove of DNA

Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein

The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models

DNMT1 and Cancer: An Electrifying Link

Aberrant epigenetic methylation is linked to the onset and progression of cancer. In this issue of Chemistry & Biology, Furst and Barton (2015) describe a sensitive electrochemical assay that can detect hyperactive epigenetic methylation in tumor tissue

Site-Directed Mutagenesis of the Cysteine Ligands to the [4Fe−4S] Cluster of Escherichia coli MutY †

International audienceThe Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7022-7025]. MutY prevents DNA mutations resulting from the misincorporation of A opposite OG by using N-glycosylase activity to remove the adenine base. An interesting feature of MutY is that it contains a [4Fe-4S] 2+ cluster that has been shown to play an important role in substrate recognition [Porello, S. L., Cannon, M. J., David, S. S. (1998) Biochemistry 37, 6465-6475]. Herein, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S] 2+ cluster of E. coli MutY with serine, histidine, and alanine. The extent to which the various mutations reduce the levels of protein overexpression suggests that coordination of the [4Fe-4S] 2+ cluster provides stability to MutY in vivo. The ability of the mutated enzymes to bind to a substrate analogue DNA duplex and their in vivo activity were evaluated. Remarkably, the effects are both substitution and position dependent. For example, replacement of cysteine 199 with histidine provides a mutated enzyme that is expressed at high levels and exhibits DNA binding and in vivo activity similar to the WT enzyme. These results suggest that histidine coordination to the iron-sulfur cluster may be accommodated at this position in MutY. In contrast, replacement of cysteine 192 with histidine results in less efficient DNA binding and in vivo activity compared to the WT enzyme without affecting levels of overexpression. The results from the site-directed mutagenesis suggest that the structural properties of the iron-sulfur cluster coordination domain are important for both substrate DNA recognition and the in vivo activity of MutY

Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases.

MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer

Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization

Diagnostic molecular markers to detect and identify primary parasitoids (Hymenoptera: Braconidae) of Ericaphis fimbriata on highbush blueberry

This research provides mitochondrial COI sequences and species-specific reverse primers for Aphidius ericaphidis Pike and Starý, Ephedrus incompletus Provencher, and Praon unicum Smith (Hymenoptera: Braconidae), which are primary parasitoids of Ericaphis fimbriata (Richards) (Hemiptera: Aphididae), an important vector of Blueberry scorch virus (BlScV) on highbush blueberry Vaccinium corymbosum L. (Ericales: Ericaceae) in the Pacific Northwest. The COI sequences are deposited under accession numbers EU574902, EU574904, GU237131, KC211020-211024, KC211027-211032.  The combination of three primers in a multiplex PCR assay detects and differentiates DNA from adults of all three parasitoid species.  When individual field-collected aphids were challenged with the multiplex PCR assay, immatures of the two numerically dominant species, A. ericaphidis and P. unicum, were readily detected as was multiparasitism by these two species.  The uncommon species, E. incompletus, was detected less frequently by multiplex PCR assay than by rearing from the aphid hosts.  The diagnostic molecular markers are useful tools for estimating of rates of parasitism and for identifying immatures of parasitoid species within aphid hosts, particularly if used in combination with rearing and dissection assays of field-collected aphids

Protein-DNA charge transport: Redox activation of a DNA repair protein by guanine radical

DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S](2+) cluster of MutY to [4Fe-4S](3+) and its decomposition product [3Fe-4S](1+). Flash/quench experiments monitored by EPR spectroscopy reveal spectra with g = 2.08, 2.06, and 2.02, characteristic of the oxidized clusters. Transient absorption spectra of poly(dGC) and [Ru(phen)(2)dppz](3+) (dppz = dipyridophenazine), generated in situ, show an absorption characteristic of the guanine radical that is depleted in the presence of MutY with formation instead of a long-lived species with an absorption at 405 nm; we attribute this absorption also to formation of the oxidized [4Fe-4S](3+) and [3Fe4S](1+) clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5'-G of a 5'-GG-3' doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With ruthenium-tethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair

NMR evidence for specific intercalation of Δ-rh(phen)_2phi^(3+) in [d(GTCGAC)_2]

The anchoring of metal complexes in the major groove of DNA through intercalation has been increasingly useful in the shape-selective design of novel metal complexes which bind DNA with high sequence-selectivity. Toward that goal, direct structural information regarding this intercalative interaction is essential. Phenanthrenequinone diimine (phi) complexes of Rh(III) bind avidly (K_b 1 ≥ 10^7) to DNA by intercalation in the major groove. Here we report the first ^1H-NMR studies of Δ-rh(phen)_2phi^(3+) bound to an oligonucleotide. These studies provide direct structural evidence for specific intercalation by this octahedral complex in the major groove of DNA

Excess maternal salt or fructose intake programmes sex-specific, stress- and fructose-sensitive hypertension in the offspring

The Western diet is typically high in salt and fructose, which have pressor activity. Maternal diet can affect offspring blood pressure, but the extent to which maternal intake of excess salt and fructose may influence cardiovascular function of the offspring is unknown. We sought to determine the effect of moderate maternal dietary intake of salt and/or fructose on resting and stimulated cardiovascular function of the adult male and female offspring. Pregnant rats were fed purified diets (±4 % salt) and water (±10 % fructose) before and during gestation and through lactation. Male and female offspring were weaned onto standard laboratory chow. From 9 to 14 weeks of age, cardiovascular parameters (basal, circadian and stimulated) were assessed continuously by radiotelemetry. Maternal salt intake rendered opposite-sex siblings with a 25-mmHg difference in blood pressure as adults; male offspring were hypertensive (15 mmHg mean arterial pressure (MAP)) and female offspring were hypotensive (10 mmHg MAP) above and below controls, respectively. Sex differences were unrelated to endothelial nitric oxide activity in vivo, but isolation-induced anxiety revealed a significantly steeper coupling between blood pressure and heart rate in salt-exposed male offspring but not in female offspring. MAP of all offspring was refractory to salt loading but sensitive to subsequent dietary fructose, an effect exacerbated in female offspring from fructose-fed dams. Circadian analyses of pressure in all offspring revealed higher mean set-point for heart rate and relative non-dipping of nocturnal pressure. In conclusion, increased salt and fructose in the maternal diet has lasting effects on offspring cardiovascular function that is sex-dependent and related to the offspring’s stress–response axis