Scientific Report 2006

Chemistry

Chemical, Biological, and Biophysical Approaches to Understanding Evolution

F.E. Romesberg, D.A. Bachovchin, J.K. Chin, R.T. Cirz, M.E. Cremeens, C. Gil-Lamaignere, N. Gingles, D.A. Harris, A.A. Henry, G.T. Hwang, A.M. Leconte, E.T. Lis, S. Matsuda, E.L. Oakman, B.A. O’Neill, T.C. Roberts, L.B. Sagle, P.A. Smith, M.C. Thielges, P. Weinkam, W. Yu, J. Zimmermann

The molecules of biology are unique because they have been evolved for function. We take a unique and multidisciplinary approach to understanding and manipulating these processes.

Increasing the Chemical and Genetic Potential of DNA

Biological information storage is based on the natural genetic alphabet, composed of the 2 base pairs guanine-cytosine and adenine-thymine. We are interested in increasing the information potential of DNA by expanding the genetic alphabet with a third base pair composed of unnatural nucleobases. Using hydrophobicity, polarity, shape complementarity, and hydrogen bonding, we are developing novel unnatural base pairs, including several that are replicable in vitro.

Nature developed the natural genetic code, not only by optimizing DNA and RNA but also by evolving the polymerases that synthesize these nucleic acids. We developed an activity-based selection system (Fig. 1) to evolve polymerases for any desired function. Using this system, we have already evolved polymerases with a variety of novel functions, including the synthesis of DNA containing one of the unnatural base pairs. We are optimizing these polymerases and evolving new ones.

Fig. 1. Activity-based phage display selection system for evolving polymerases with novel activity. Infection of phage (B) with the polymerase library (A) leads to production of phage particles that display 0–1 copies of the polymerase and 3–5 copies of the acidic peptide. Phage particles are combined with DNA primer–template (C) and incubated with the desired nucleoside triphosphates. Active mutants are isolated (D) and characterized.

DNA Damage Response

Evolution requires mutation, but mutations also make cells susceptible to aging and cancer. It is now understood that at times of sufficient stress, cells induce error-prone replication to facilitate their own evolution. We used genome-wide high-throughput methods to identify genes involved in both error-free and error-prone responses to DNA stress in budding yeast. Characterization of the proteins required for mutation in mammalian cells will revolutionize our understanding of cancer and aging and identify drug targets whose inhibition might actually inhibit these processes.

In bacteria, induced mutation can lead to antibiotic resistance. We have fully characterized the mechanism of induced mutation and antibiotic resistance in Escherichia coli (Fig. 2), and we are characterizing these pathways in other bacterial pathogens. We are also designing a drug that inhibits bacterial mutation and thus evolution.

Fig. 2. Induced mutation in E coli is controlled by the transcriptional regulator LexA. Duplex DNA is shown in black; each gray box indicates a double-strand break. The breaks can be repaired through pathway A, B, or C. Derepression of the error-prone polymerases Pol IV and Pol V leads to mutations (pathway D).

Evolution of Protein Dynamics

The products of evolution are molecules with unique vibrational dynamics. The study of vibrational dynamics in proteins and nucleic acids has been limited by spectral complexity, but selective deuteration of a protein or a nucleic acid results in a carbon-deuterium oscillator that absorbs light in an otherwise transparent region of the infrared spectrum. The synthesis of selectively deuterated proteins has provided us with a residue-specific probe of flexibility, function, and folding. We are also using multidimensional femtosecond spectroscopy to characterize how protein motion is evolved during the somatic evolution of antibodies. We discovered that the immune system can manipulating protein dynamics, a finding that suggests a role for these dynamics in molecular recognition.

Publications

Chin, J.K., Bashkirov, V.I., Heyer, W.D., Romesberg, F.E. Esc4/Rtt107 and the control of recombination during replication. DNA Repair (Amst.) 5:618, 2006.

Cirz, R.T., Gingles, N., Romesberg, F.E. Side effects may include evolution. Nat. Med. 8:890, 2006.

Cirz, R.T., O’Neill, B.M., Hammond, J.A., Head, S.R., Romesberg, F.E. Defining the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin. J. Bacteriol. 188:7101, 2006.

Cirz, R.T., Romesberg, F.E. Induction and inhibition of ciprofloxacin resistance-conferring mutations in hypermutator bacteria. Antimicrob. Agents Chemother. 50:220, 2006.

Cremeens, M., Fujisaki, H., Zhang, Y., Zimmermann, J., Sagle, L.B., Matsuda, S., Dawson, P.E., Straub, J.E., Romesberg, F.E. Efforts toward developing direct probes of protein dynamics. J. Am. Chem. Soc. 128:6028, 2006.

Dupradeau, F.-Y., Case, D.A., Yu, C., Jimenez, R., Romesberg, F.E. Differential solvation and tautomer stability of a model base pair within the minor and major grooves of DNA. J. Am. Chem. Soc. 127:15612, 2005.

Henry, A.A., Romesberg, F.E. Evolution of DNA polymerases with novel activities. Curr. Opin. Biotechnol. 16:370, 2005.

Hwang, G.T., Romesberg, F.E. Substituent effects on the pairing and polymerase recognition of simple unnatural base pairs. Nucleic Acids Res. 34:2037, 2006.

Kim, Y., Leconte, A.M., Hari, Y., Romesberg, F.E. Stability and polymerase recognition of pyridine nucleobase analogues: role of minor-groove H-bond acceptors. Angew. Chem. Int. Ed., in press.

Leconte, A.M., Chen, L., Romesberg, F.E. Polymerase evolution: efforts toward expansion of the genetic code. J. Am. Chem. Soc. 127:12470, 2005.

Leconte, A.M., Matsuda, S., Hwang, G., Romesberg, F.E. Efforts towards expansion of the genetic alphabet: pyridone and methyl pyridone nucleobases. Angew. Chem. Int. Ed. 45:4326, 2006.

Leconte, A.M., Matsuda, S., Romesberg, F.E. An efficiently extended class of unnatural base pairs. J. Am. Chem. Soc. 128:6780, 2006.

Leconte, A.M., Romesberg, F.E. Amplify this! DNA and RNA get a third base pair. Nat. Methods 3:667, 2006.

Lis, E.T., Romesberg, F.E. Role of Doa1 in the Saccharomyces cerevisiae DNA damage response. Mol. Cell. Biol. 26:4122, 2006.

Matsuda, S., Henry, A.A., Romesberg, F.E. Optimization of unnatural base pair packing for polymerase recognition. J. Am. Chem. Soc. 128:6369, 2006.

Sagle, L.B., Zimmermann, J., Dawson, P.E., Romesberg, F.E. Direct and high-resolution characterization of cytochrome c equilibrium folding. J. Am. Chem. Soc., in press.

Sagle, L.B., Zimmermann, J., Matsuda, S., Dawson, P.E., Romesberg, F.E. Redox-coupled dynamics and folding in cytochrome c. J. Am. Chem. Soc. 128:7909, 2006.

Zimmermann, J., Oakman, E.L., Thorpe, I.F., Shi, X., Abbyad, P., Brooks, C.L. III, Boxer, S.G., Romesberg, F.E. Antibody evolution constrains conformational hetereogeneity by tailoring protein dynamics. Proc. Natl. Acad. Sci. U. S. A. 103:13722, 2006.