Brian S. DeDecker

Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA‐binding activity of such mutants to understand the mechanism of second‐site suppressor mutations. Double‐mutant cycles show that N239Y and N268D act as ‘global stability’ suppressors by increasing the stability of the cancer mutants G245S and V143A—the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.

Localization of the retinal protonated Schiff base counterion in rhodopsin.

Semiempirical molecular orbital calculations are combined with 13C NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C10 to C15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed 13C NMR data of rhodopsin exhibit downfield chemical shifts from C8 to C13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., I. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods. These data constrain the location and the orientation of the Glu113 side chain, which is known to be the counterion in rhodopsin, and argue for a strong interaction centered at C12 of the retinylidene chain.

Allosteric drugs: thinking outside the active-site box.

Recently, a class of small molecules that thermally stabilize the tumor suppressor p53 was selected from a small-molecule library. This, and other recent work, demonstrates the feasibility of taking a lead from nature and selecting new classes of drugs that function by allosteric mechanisms.

Modifiers of notch transcriptional activity identified by genome-wide RNAi

BackgroundThe Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription.ResultsEmploying complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components.ConclusionsThe genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.

Gene and library synthesis without amplification: polymerase step reaction (PSR).

Current gene synthesis methods often incorporate a PCR amplification step in order to yield final material sufficient for resolution from multiple off-products. These amplification steps can cause stochastic sampling effects that propagate errors in gene synthesis or decrease variability when applied to the construction of randomized libraries. We have developed a simple DNA polymerase-based gene synthesis reaction, polymerase step reaction (PSR), that assembles DNA oligonucleotides in a unidirectional fashion without the need for amplification. We demonstrate that PSR is efficient, with little off-product production, no detectable error propagation, and maximized variability in the synthesis of a phage display library.

The Polymerase Step Reaction (PSR) Method for Gene and Library Synthesis.

Current gene synthesis methods often incorporate a PCR-amplifying step in order to yield sufficient final product that is detectable and resolvable from multiple off-products. This amplification step can cause stochastic sampling effects that propagate errors during the synthesis and lower the variability when applied towards the construction of randomized libraries. We present the method for polymerase step reaction (PSR), a simple DNA polymerase-based gene synthesis reaction that assembles DNA oligonucleotides in a unidirectional fashion without the need for a PCR-type amplification (Lee et al., BioTechniques 59:163-166, 2015). The PSR method is simple and efficient with little off-product production, undetected stochastic sampling effects, and maximized variability when used to synthesize phage display libraries.