Rapid photoinduced electron transfer is demonstrated over a distance of greater than 40 angstroms between metallointercalators that are tethered to the 5' termini of a 15-base pair DNA duplex. An oligomeric assembly was synthesized in which the donor is Ru(phen)2dppz2+ (phen, phenanthroline, and dppz, dipyridophenazine) and the acceptor is Rh(phi)2phen3+ (phi, phenanthrenequinone diimine). These metal complexes are intercalated either one or two base steps in from the helix termini. Although the ruthenium-modified oligonucleotide hybridized to an unmodified complement luminesces intensely, the ruthenium-modified oligomer hybridized to the rhodium-modified oligomer shows no detectable luminescence. Time-resolved studies point to a lower limit of 10(9) per second for the quenching rate. No quenching was observed upon metallation of two complementary octamers by Ru(phen)3(2+) and Rh(phen)3(3+) under conditions where the phen complexes do not intercalate. The stacked aromatic heterocycles of the DNA duplex therefore serve as an efficient medium for coupling electron donors and acceptors over very long distances.

Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, R~( b p y )~d p p z~+ and Ru(phen)zdppz2+ (bpy = 2,2'-bipyridine; phen = 1,lO-phenanthroline; dppz = dipyrido[3,2:a-2',3':~]-phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)zdppz2+ and Ru(phen)2dppzz+ are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.Considerable attention has been given to the design of small molecules that bind to DNA with site selectivity so as to develop novel therapeutics and chemical probes for nucleic acid sites and structures, as well as novel diagnostic agents targeted to double-helical DNA (Pyle & Dervan, 1986;Moser & Dervan, 1987;Hecht, 1986;Tullius, 1988). Our laboratory has focused in part on the development of transitionmetal complexes as probes of nucleic acid structure (Chow & Barton, 1992;Mei & Barton, 1986;Kirshenbaum et al., 1988;Barton, 1986). We have found that ruthenium complexes serve as very sensitive luminescent reporters of DNA in aqueous solution and may become particularly useful in developing new diagnostics.Ruthenium complexes are ideally suited for application as sensitive noncovalent probes for polymer structure. The complexes are water-soluble, coordinatively saturated, and inert to substitution. Polypyridyl complexes of ruthenium-(11) furthermore are intensely colored owing to a wellcharacterized, localized metal-to-ligand charge transfer (MLCT) transition (Juris et al., 1988). Importantly, this transition is perturbed on binding to DNA, providing a sensitive spectroscopic handle for interactions with nucleic acids (Pyle et al., 1989). Tris( 1 ,IO-phenanthroline)ruthenium(II), Ru-(phen)j2+, has been established, primar...

HIV-1 is able to infect nondividing cells productively in part because the postentry viral nucleoprotein complexes are actively imported into the nucleus. In this manuscript, we identify a novel nuclear localization signal (NLS) in the viral integrase (IN) protein that is essential for virus replication in both dividing and nondividing cells. The IN NLS stimulates the efficient nuclear accumulation of viral DNA as well as virion-derived IN protein during the initial stages of infection but is dispensable for catalytic function. Because this NLS is required for infection irrespective of target cell proliferation, we suggest that interactions between uncoated viral nucleoprotein complexes and the host cell nuclear import machinery are critical for HIV-1 infection of all cells.

The bimolecular quenching of the metal-to-ligand charge transfer (MLCT) excited state of Ru(phen)z-(dppz)2+ (phen = 1,lO-phenanthroline, dppz = dipyrido[3,2-a:2',3'-clphenazine) by proton transfer has been investigated in homogeneous acetonitrile solutions and in the presence of calf thymus DNA. In acetonitrile the monoexponential decay of the MLCT excited state emission of R~(phen)2(dppz)~+ is dynamically quenched by proton donors with pKa = 4.7-15.7. The emission lifetimes and quenching when the complex is bound to DNA have been measured for racemic mixtures, as well as A-and A-R~(phen)2(dppz)~+, and the values compared. In the presence of DNA the biexponential decay of the emission is quenched dynamically and three times slower than in acetonitrile when the quencher, in this case hydroquinone, is hydrophilic. Static quenching is observed in the presence of DNA when a hydrophobic proton donor, o-chlorophenol, is utilized. The static quenching with o-chlorophenol is shown to arise solely from the quenching of the long-lived component. These observations are explained in terms of two different modes of binding between the complex and DNA, as well as the different affinities for the aqueous medium of the quenchers.

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

There has been considerable interest in the development of new chemical methods to distinguish and detect nucleic acids with sequence specificity.1 One focus of our laboratory has been on the application of ruthenium complexes2,3 as spectroscopic probes

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both 13C-palmitate and 13C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.