Widespread transient Hoogsteen base pairs in canonical duplex DNA with variable energetics.

Hoogsteen (HG) base pairing involves a 180° rotation of the purine base relative to Watson-Crick (WC) base pairing within DNA duplexes, creating alternative DNA conformations that can play roles in recognition, damage induction and replication. Here, using nuclear magnetic resonance R1ρ relaxation dispersion, we show that transient HG base pairs occur across more diverse sequence and positional contexts than previously anticipated. We observe sequence-specific variations in HG base pair energetic stabilities that are comparable with variations in WC base pair stability, with HG base pairs being more abundant for energetically less favourable WC base pairs. Our results suggest that the variations in HG stabilities and rates of formation are dominated by variations in WC base pair stability, suggesting a late transition state for the WC-to-HG conformational switch. The occurrence of sequence and position-dependent HG base pairs provide a new potential mechanism for achieving sequence-dependent DNA transactions.

A historical account of Hoogsteen base-pairs in duplex DNA.

In 1957, a unique pattern of hydrogen bonding between N3 and O4 on uracil and N7 and N6 on adenine was proposed to explain how poly(rU) strands can associate with poly(rA)-poly(rU) duplexes to form triplexes. Two years later, Karst Hoogsteen visualized such a noncanonical A-T base-pair through X-ray analysis of co-crystals containing 9-methyladenine and 1-methylthymine. Subsequent X-ray analyses of guanine and cytosine derivatives yielded the expected Watson-Crick base-pairing, but those of adenine and thymine (or uridine) did not yield Watson-Crick base-pairs, instead favoring "Hoogsteen" base-pairing. More than two decades ensued without experimental "proof" for A-T Watson-Crick base-pairs, while Hoogsteen base-pairs continued to surface in AT-rich sequences, closing base-pairs of apical loops, in structures of DNA bound to antibiotics and proteins, damaged and chemically modified DNA, and in polymerases that replicate DNA via Hoogsteen pairing. Recently, NMR studies have shown that base-pairs in duplex DNA exist as a dynamic equilibrium between Watson-Crick and Hoogsteen forms. There is now little doubt that Hoogsteen base-pairs exist in significant abundance in genomic DNA, where they can expand the structural and functional versatility of duplex DNA beyond that which can be achieved based only on Watson-Crick base-pairing. Here, we provide a historical account of the discovery and characterization of Hoogsteen base-pairs, hoping that this will inform future studies exploring the occurrence and functional importance of these alternative base-pairs.

Probing transient Hoogsteen hydrogen bonds in canonical duplex DNA using NMR relaxation dispersion and single-atom substitution.

Nucleic acids transiently morph into alternative conformations that can be difficult to characterize at the atomic level by conventional methods because they exist for too little time and in too little abundance. We recently reported evidence for transient Hoogsteen (HG) base pairs in canonical B-DNA based on NMR carbon relaxation dispersion. While the carbon chemical shifts measured for the transient state were consistent with a syn orientation for the purine base, as expected for A(syn)•T(anti) and G(syn)•C(+)(anti) HG base pairing, HG type hydrogen bonding could only be inferred indirectly. Here, we develop two independent approaches for directly probing transient changes in N-H···N hydrogen bonds and apply them to the characterization of transient Hoogsteen type hydrogen bonds in canonical duplex DNA. The first approach takes advantage of the strong dependence of the imino nitrogen chemical shift on hydrogen bonding and involves measurement of R(1ρ) relaxation dispersion for the hydrogen-bond donor imino nitrogens in G and T residues. In the second approach, we assess the consequence of substituting the hydrogen-bond acceptor nitrogen (N7) with a carbon (C7H7) on both carbon and nitrogen relaxation dispersion data. Together, these data allow us to obtain direct evidence for transient Hoogsteen base pairs that are stabilized by N-H···N type hydrogen bonds in canonical duplex DNA. The methods introduced here greatly expand the utility of NMR in the structural characterization of transient states in nucleic acids.

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2.

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by ß-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.