Hoogsteen base-pairing involves a 180 degree rotation of the purine base relative to Watson-Crick 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 Hoogsteen base-pairs occur across more diverse sequence and positional contexts than previously anticipated. We observe sequence-specific variations in Hoogsteen base-pair energetic stabilities that are comparable to variations in Watson-Crick base-pair stability, with Hoogsteen base-pairs being more abundant for energetically less favorable Watson-Crick base-pairs. Our results suggest that the variations in Hoogsteen stabilities and rates of formation are dominated by variations in Watson-Crick base pair stability, suggesting a late transition state for the Watson-Crick to Hoogsteen conformational switch. The occurrence of sequence and position-dependent Hoogsteen base-pairs provide a new potential mechanism for achieving sequence-dependent DNA transactions.
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 non-canonical 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 exists 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.
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 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 R1ρ 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.
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 -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 G␣ 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 pleckstrinhomology 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 domainmediated regulation of GPCRs beyond G␣ q -coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.It has been classically described that G protein-coupled receptor (GPCR) 2 desensitization involves rapid receptor phosphorylation by second messenger-dependent kinases and/or G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes arrestin binding to the receptor followed by GPCR internalization in clathrin-coated pits. Therefore, the paradigmatic GRK major function is to enable arrestin binding to the receptor and uncoupling from the heterotrimeric G protein (1). However, GPCR signaling may also be specifically attenuated not only at the receptor level but also at the G protein level (2). In recent years, the intracellular regulator of G protein signaling (RGS) proteins have been discovered to serve additional, mostly negative modulatory roles, in G protein-mediated signal transduction (3). More than 30 proteins containing RGS or RGS homology (RH) domains have been described so far (4). Most of these proteins interact with G␣ i or G␣ q subunits, but there is also evidence for the interaction of RGS2 and the truncated form of RGS3 with the G␣ s subunit (5, 6). The seven GRKs proteins described so far contain an N-terminal RGS-homology domain (RH), a catalytic central...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.