Aminolysis of 2′-Deoxyinosine Aryl Ethers: Nucleoside Model Studies for the Synthesis of Functionally Tethered Oligonucleotides

Abstract

Several O⁶-aryl-2′-deoxyinosines were synthesized and found to undergo conversion to N⁶-substituted-2′-deoxyadenosines upon treatment with aqueous amines. The kinetics for reaction of these nucleosides with various amines suggests that O⁶-phenyl- and O⁶-(p-nitrophenyl)-2′-deoxyinosine are suitable “convertible nucleoside” precursors for the site-specific introduction of functionally tethered 2′-deoxyadenosines into DNA.     

Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin metathesis

This protocol provides a detailed procedure for the preparation of stapled α-helical peptides, which have proven their potential as useful molecular probes and as next-generation therapeutics. Two crucial features of this protocol are (i) the construction of peptide substrates containing hindered α-methyl, α-alkenyl amino acids and (ii) the ring-closing olefin metathesis (RCM) of the resulting resin-bound peptide substrates. The stapling systems described in this protocol, namely bridging one or two turns of an α-helix, are highly adaptable to most peptide sequences, resulting in favorable RCM kinetics, helix stabilization and promotion of cellular uptake.     

Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function

Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in?vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.     

Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1

A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility.     

Subunit-specific protein footprinting reveals significant structural rearrangements and a role for N-terminal Lys-14 of HIV-1 Integrase during viral DNA binding

To identify functional contacts between HIV-1 integrase (IN) and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys- and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The foot-printing studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes.     

Structural and Biochemical Analysis of DNA Helix Invasion by the Bacterial 8-Oxoguanine DNA Glycosylase MutM

MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe¹¹⁴ and Met⁷⁷ in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met⁷⁷ plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe¹¹⁴, on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair.