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Research in the Wuttke lab spans two main areas, telomere biology and plasticity in binding.听 Details on projects in these areas can be found at our lab web page,听

Telomere Biology

Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes that are essential for chromosome stability and cellular proliferation. Telomeric DNA does not encode for proteins, instead it consists of tandem repeats of TG-rich sequences of double-stranded DNA that terminate in a 3垄 single-stranded DNA overhang. Protection of this overhang is essential. When left unprotected, this overhang initiates DNA damage responses that lead to catastrophic events permanently damaging the genome and resulting in apoptosis or senescence. Furthermore, telomere shortening due to the inability of the DNA-replication machinery to fully replicate the ends is a critical mechanism of tumor suppression as well as a hallmark of aging. Continually proliferating cells maintain adequate telomeres through the action of the reverse transcriptase telomerase.Telomeres are important to human health because dysregulation of either telomere protection or telomerase activity causes many human diseases. Notably, over 90% of human cancers activate telomerase for continued proliferation.

Our research in this area aims to understand how telomere-associated proteins protect and maintain telomeres. Key questions include how subunits of the telomerase enzyme contribute to activity, how the single-strand DNA overhang is shielded from the DNA-damage machinery, and whether capping activity also regulates听telomerase action. We develop this knowledge by first understanding the core activities of key telomere factors, then testing these activities in a reconstituted telomerase assay and validating our knowledge directly in the organism.

Plasticity in Molecular Recognition

Many biologically critical recognition events involve the specific binding of flexible ligands such as single-stranded (ss) DNA, RNA, peptides and carbohydrates. Structural plasticity, defined as the ability of an interface to adopt alternate conformations when bound to different ligands, has been invoked to explain binding specificity and promiscuity in several protein/ligand systems. Furthermore, an understanding of the malleability of a binding interface is increasingly recognized as key to predicting its binding activity and specificity. Discerning the scope and mechanisms of rearrangements at binding interfaces is essential to understanding the biophysics of molecular recognition events. The focus of this proposal is to investigate the extent of structural plasticity in the recognition of these flexible ligands.

We use the recognition of ssDNA by the telomere end-binding proteins as the predominant model to characterize the contribution of structural plasticity to recognition. The telomere-end binding proteins Pot1 and Cdc13 bind the conserved 3鈥� ssDNA overhang at telomeres. This binding is required for cellular viability. However, the sequence of the overhang is somewhat variable, meaning that these proteins need to bind divergent ligands while maintaining exquisite specificity. Extensive evidence suggests that the protein/nucleic acid interface adopts altered configurations in the presence of different ligands that bind with similar affinities. We are investigating the hypothesis that this structural plasticity is important for specificity. Moreover, the malleability of the interface may further contribute to function by providing a way to physically alter the structure and accessibility of the 3鈥� end. We us an integrated set of strategies to address this question, ranging from determination of high-resolution structures to in vivo assessment of activities.

K. A. Lewis, D. A. Pfaff, J. N. Earley, S. E. Altschuler, and D. S. Wuttke, 鈥淭he Tenacious Recognition of Yeast Telomere Sequence by Cdc13 is Fully Exerted by a Single OB-Fold Domain,鈥澨�Nucleic Acids Res.,听2013, Sept 20 epub ahead of print

T. H. Dickey, S. E. Altschuler and D. S. Wuttke, 鈥溌璖ingle-stranded DNA-binding Proteins: Multiple Domains for Multiple Functions,鈥澨�Structure,听2013,听21, 1074-84

T. H. Dickey, M. A. McKercher, and D. S.听Wuttke,听鈥�,鈥澨�Structure,听2013,听21, 121-32

S. E. Altschuler, J. E. Croy, and D. S. Wuttke, 鈥淎 Small Molecule Inhibitor of the听S. pombe听Pot1 binding to telomeric DNA,鈥澨�Biochemistry,听2012,听51, 7833-45

K. A. Lewis and D. S. Wuttke, 鈥淭elomerase and Telomere-Associated Proteins: Structural insights into mechanism and evolution,鈥澨�Structure,听2012,听20, 28 - 39

S. E. Altschuler, T. H. Dickey, and D. S. Wuttke, 鈥溾�澨�Biochemistry,听2011,听50, 7503-15

E. K. Mandell, A. D. Gelinas, D. S. Wuttke, V. Lundblad, 鈥溙�Biochemistry,听2011,听50,听6289-91

J. Lee, E. K. Mandell, T. Rao, D. S. Wuttke and V. Lundblad, 鈥淚nvestigating the role of the Est3 protein in yeast telomere replication,鈥澨�Nucleic Acids Res.,听2010,听38, 2279-2290

A. D. Gelinas, M. Paschini, F. E. Reyes, A. H茅roux, R. T. Batey, V. Lundblad and D. S. Wuttke, 鈥淭elomere capping proteins are structurally related to RPA with an additional telomere-specific domain鈥澨�Proc. Natl. Acad. Sci. USA,听2009,听106, 19298-19303

J. E. Croy and D. S. Wuttke, 鈥淚nsights into the dynamics of specific telomeric single-stranded DNA recognition by Pot1pN,鈥澨�J. Mol. Biol.,听2009,听387, 935-938

J. E. Croy, S. E. Altschuler, N. E. Grimm, and D. S. Wuttke, 鈥淣onadditivity in the recognition of single-stranded DNA by the Schizosaccharomyces pombe protection of telomeres 1 DNA-binding domain, Pot1-DBD,鈥澨�Biochemistry,听2009,听48, 6864-6875

D. C. Zappulla, J. N. Roberts, K. Goodrich, T. R. Cech, and D. S. Wuttke, 鈥淚nhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p,鈥澨�Nucleic Acid Res.,听2009,听37, 354-367

J. E. Croy, J. L. Fast, N. E. Grimm, and听D. S. Wuttke, 鈥淭hermodynamic characterization of specific single-stranded telomeric DNA binding to the听S. pombe, Protection of telomeres 1 (POT1), protein,听Biochemistry,听2008,听47, 4345-4358

A. E. Eldridge and D. S. Wuttke, 鈥淭he mechanism of recognition of ssDNA by the Cdc13-DBD,鈥澨�Nucleic Acid Res.,听2008,听36, 1624-33

D. L. Theobald and D. S. Wuttke, 鈥淎ccurate structural correlations from maximum likelihood superpositions,鈥澨�PLOS Comp. Biology,听2008,听e43

J. R. Croy and D. S. Wuttke, 鈥淭hemes in ssDNA recognition by telomere end-protection proteins,鈥澨�TIBS,听2006,听31, 516-525

D. L. Theobald and D. S. Wuttke, THESEUS: Maximum likelihood superpositioning and analysis of macromolecular structures,听Bioinformatics,听2006,听22, 2171-2

A. M. Eldridge, W. A. Halsey, and D. S Wuttke, 鈥淚dentification of the determinants for the specific recognition of single-strand telomeric DNA by Cdc13,鈥澨�Biochemistry,听2005, 45, 871-879

D. L. Theobald and D. S. Wuttke, 鈥淒ivergent evolution within protein superfolds inferred from profile-based phylogenetics鈥澨�J. Mol. Biol.,听2005,听354, 722-737

D. L. Theobald and D. S. Wuttke, 鈥淧rediction of multiple tandem OB-folds in telomere end-binding proteins Pot1 and Cdc13鈥澨�Structure,听2004,听12, 1877-1879.

R. M. Mitton-Fry, E. M. Anderson, L. W. Glustrom, D. L. Theobald and D. S. Wuttke, 鈥淪tructural basis for telomeric single-stranded DNA recognition by yeast Cdc13,鈥澨�J. Mol. Biol.,听2004,听338, 241-255.

Daniel M. Strauss, Leslie W. Glustrom, and Deborah S. Wuttke. "Towards an understanding of the poliovirus replication complex: the solution structure of the soluble domain of the poliovirus 3A protein,"听J. Mol. Biol., 330, 225-234 (2003).

Douglas L. Theobald, Rachel M. Mitton-Fry, and Deborah S. Wuttke. "Nucleic acid recognition by OB-fold proteins,"听Annu. Rev. Biophys. and Biomol. Struct., Feb. 18 (2003).

Emily M. Anderson, and Deborah S. Wuttke. "Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13,"听Biochemistry, 42, 3751-3758 (2003).

Deanna Dahlke Ojennus, Sarah E. Lehto, and Deborah S. Wuttke. "Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments,"听Protein Science, 12, 44-55 (2003).

Leslie W. Glustrom, Rachel M. Mitton-Fry, and Deborah S. Wuttke. "Environmental estrogens and breast cancer: the importance of hydroxylated intermediates,"听J. Natl. Cancer Institute, 94, 68-69 (2002).

Emily M. Anderson, Wayne A. Halsey, and Deborah S. Wuttke. "Delineation of the high-affinity single-stranded telomeric DNA-binding domain of听S. cerevisiae听Cdc13,"听Nucleic Acids Research, 30, 4305-4313 (2002).

Rachel M. Mitton-Fry, and Deborah S. Wuttke. "1H,听13C and听15N resonance assignments of the DNA-binding domain of the essential protein Cdc13 complexed with single-stranded telomeric DNA,"听J. Biomol. NMR, 22, 379-380 (2002).

Rachel M. Mitton-Fry, Emily M. Anderson, Timothy T. Hughes, Victoria Lundblad, and Deborah S. Wuttke. "Conserved structure for single-stranded relomeric DNA recognition,"听Science, 296, 145-147 (2002).