Repair of oxidatively damaged guanine in Saccharomyces cerevisiae by an alternative pathway

AbstractBackground: Transversion mutations are caused by 8-oxoguanine (OG), a DNA lesion produced by the spontaneous oxidation of guanine nucleotides, which mis-pairs with adenine during replication. Resistance to this mutagenic threat is mediated by the GO system, the components of which are functionally conserved in bacteria and mammals. To date, only one of three GO system components has been identified in the budding yeast Saccharomyces cerevisiae, namely the OG:C-specific glycosylase/lyase yOgg1. Furthermore, S. cerevisiae has been reported to contain a unique glycosylase/lyase activity, yOgg2, which excises OG residues opposite adenines. Paradoxically, according to the currently accepted model, yOgg2 activity should increase the mutagenicity of OG lesions. Here we report the isolation of yOgg2 and the elucidation of its role in oxidative mutagenesis.Results: Borohydride-dependent cross-linking using an OG-containing oligonucleotide substrate led to the isolation of yOgg1 and a second protein, Ntg1, which had previously been shown to process oxidized pyrimidines in DNA. We demonstrate that Ntg1 has OG-specific glycosylase/lyase activity indistinguishable from that of yOgg2. Targeted disruption of the NTG1 gene resulted in complete loss of yOgg2 activity and yeast lacking NTG1 had an elevated rate of A:T to C:G transversions.Conclusions: The Ntg1 and yOgg2 activities are encoded by a single gene. We propose that yOgg2 has evolved to process OG:A mis-pairs that have arisen through mis-incorporation of 8-oxo-dGTP during replication. Thus, the GO system in S. cerevisiae is fundamentally distinct from that in bacteria and mammals

In vitro selection of RNA aptamers against a composite small molecule-protein surface

A particularly challenging problem in chemical biology entails developing systems for modulating the activity of RNA using small molecules. One promising new approach towards this problem exploits the phenomenon of ‘surface borrowing,’ in which the small molecule is presented to the RNA in complex with a protein, thereby expanding the overall surface area available for interaction with RNA. To extend the utility of surface borrowing to include potential applications in synthetic biology, we set out to create an ‘orthogonal’ RNA-targeting system, one in which all components are foreign to the cell. Here we report the identification of small RNA modules selected in vitro to bind a surface-engineered protein, but only when the two macromolecules are bound to a synthetic bifunctional small molecule

Inhibition of Oncogenic Wnt Signaling through Direct Targeting of β-Catenin

Aberrant activation of signaling by the Wnt pathway is strongly implicated in the onset and progression of numerous types of cancer. Owing to the persistent dependence of these tumors on Wnt signaling for growth and survival, inhibition of this pathway is considered an attractive mechanism-based therapeutic approach. Oncogenic activation of Wnt signaling can ensue from a variety of distinct aberrations in the signaling pathway, but most share the common feature of causing increased cellular levels of β-catenin by interfering with its constitutive degradation. β-Catenin serves as a central hub in Wnt signaling by engaging in crucial protein–protein interactions with both negative and positive effectors of the pathway. Direct interference with these protein–protein interactions is a biologically compelling approach toward suppression of β-catenin hyperactivity, but such interactions have proven intransigent with respect to small-molecule targeting. Hence β-catenin remains an elusive target for translational cancer therapy. Here we report the discovery of a hydrocarbon-stapled peptide that directly targets β-catenin and interferes with its ability to serve as a transcriptional coactivator for T-cell factor (TCF) proteins, the downstream transcriptional regulators of the Wnt pathway.Chemistry and Chemical BiologyStem Cell and Regenerative Biolog

All-Atom Model for Stabilization of α-Helical Structure in Peptides by Hydrocarbon Staples

Recent work has shown that the incorporation of an all-hydrocarbon “staple” into peptides can greatly increase their α-helix propensity, leading to an improvement in pharmaceutical properties such as proteolytic stability, receptor affinity and cell-permeability. Stapled peptides thus show promise as a new class of drugs capable of accessing intractable targets such as those that engage in intracellular protein-protein interactions. The extent of α-helix stabilization provided by stapling has proven to be substantially context dependent, requiring cumbersome screening to identify the optimal site for staple incorporation. In certain cases, a staple encompassing one turn of the helix (attached at residues i and i+4) furnishes greater helix stabilization than one encompassing two turns (i,i+7 staple), which runs counter to expectation based on polymer theory. These findings highlight the need for a more thorough understanding of the forces that underlie helix stabilization by hydrocarbon staples. Here we report all-atom Monte Carlo folding simulations comparing unmodified peptides derived from RNAse A and BID BH3 with various i,i+4 and i,i+7 stapled versions thereof. The results of these simulations were found to be in quantitative agreement with experimentally determined helix propensities. We also discovered that staples can stabilize quasi-stable decoy conformations, and that the removal of these states plays a major role in determining the helix stability of stapled peptides. Finally, we critically investigate why our method works, exposing the underlying physical forces that stabilize stapled peptides.Chemistry and Chemical Biolog

A DESIGNED INHIBITOR OF BASE-EXCISION DNA-REPAIR

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Structure of a DNA glycosylase searching for lesions

DNA glycosylases must interrogate millions of base pairs of undamaged DNA in order to locate and then excise one damaged nucleobase. The nature of this search process remains poorly understood. Here we report the use of disulfide cross-linking (DXL) technology to obtain structures of a bacterial DNA glycosylase, MutM, interrogating undamaged DNA. These structures, solved to 2.0 angstrom resolution, reveal the nature of the search process: The protein inserts a probe residue into the helical stack and severely buckles the target base pair, which remains intrahelical. MutM therefore actively interrogates the intact DNA helix while searching for damage. One of the most formidable needle-in-ahaystack challenges in biology is that faced by DNA glycosylases as they locate and excise damaged DNA nucleobases embedded in a greater than millionfold excess of undamaged DNA (1, 2). The challenge is compounded by the fact that many damaged bases targeted by DNA glycosylases are only subtl