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Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

D.A. Malyshev, K. Dhami, H.T. Quach, T. Lavergne, P. Ordoukhanian, A. Torkamani, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2012) 109:12005-12010.
pubpic2012malyshevWe examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs and demonstrate that this DNA may be amplified with high efficiency and with greater than 99.9% fidelity. The results demonstrate that for PCR and PCR-based applications, d5SICS-ddNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, provides the first fully functional six-letter genetic alphabet. Read more in Nature and Biotechniques.

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The mechanism of action of the arylomycin antibiotics and the effects of signal peptidase I inhibition

P.A. Smith, F.E. Romesberg, Antimicrob. Agents Chemother. (2012) 56:5054-5060.
pubpic2012smithWe examine arylomycin activity as a function of concentration, bacterial cell density, target expression levels, and bacterial growth phase. We find that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. We also demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.

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Protein dynamics and the diversity of the antibody response

R. Adhikary, W. Yu, M. Oda, J. Zimmermann, F.E. Romesberg, J. Biol. Chem. (2012) 287:27139-27147.
pubpic2012adhikaryWe report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight antibodies (Abs) elicited to a chromophoric antigen (MPTS). Three of the Abs arose from unique germline Abs, while the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. By characterizing both flexibility and conformational heterogeneity, we show that point mutations are capable of fixing significant differences in protein dynamics.

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Yeasts acquire resistance secondary to antifungal drug treatment by adaptive mutagenesis

D. Quinto-Alemany, A. Canerina-Amaro, L.G. Hernández-Abad, F. Machín, F.E. Romesberg, C. Gil-Lamaignere, PLoS ONE (2012) 7:e42279.
pubpic2012quinto_alemanyStressful lifestyle associated mutation (SLAM) assays are used to demonstrate that both S. cerevisiae and C. albicans undergo mutations to acquire resistance to the antifungal agents 5-fluorocytosine and caspofungin. Examination of the mutation spectrum shows that these aquired mutations are different from those that the occur spontaneously, in the absence of antifungal treatment. Overall, the work present a much needed model system for studying adaptive mutagenesis in eukaryotes.

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KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

K. Betz, D.A. Malyshev, T. Lavergne, W. Welte, K. Diederichs, T.J. Dwyer, P. Ordoukhanian, F.E. Romesberg, A. Marx, Nat. Chem. Biol. (2012) 8:612-614.
pubpic2012betzWe report crystal structures of KlenTaq DNA polymerase at different stages of replicating dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure. Read more in Live Science.

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Carbon-deuterium bonds as probes of protein thermal unfolding

W. Yu, P. Dawson, J. Zimmermann, F.E. Romesberg, J. Phys. Chem. B (2012) 116:6397-6403.
pubpic2012yuWe report a residue-specific characterization of the thermal unfolding mechanism of cyt c using C-D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, are highlights of the utility of the C-D technique.