News and Publications

Structure and Dynamics of DNA Intermediates and Processing Enzymes Involved in Genetic Recombination and Repair

W.J. Chazin, D.P. Millar, A. Edwards,* D. Gonzales, M. Kainosho,** W.-C. Lam, S. Mangahas, G. Mer, A. Ono,** A.G. Palmer III,*** S. Parikh, N. Seeman,**** J.A. Tainer

* University of Toronto, Toronto, Canada
** Tokyo Metropolitan University, Tokyo, Japan
*** Columbia University, New York, NY
**** New York University, New York, NY

The goal of our multidisciplinary research program is to understand the structural biology of unusual DNA structures and the processing of these structures by the genetic recombination and repair machinery of the cell. Branched and modified DNA structures are formed during routine events of genetic recombination and as a by-product of chemical and radiation damage. Our efforts to date have focused on a specific 4-arm DNA crossover structure, the Holliday junction, which is formed as a transient intermediate during genetic recombination and repair. The structure and flexibility of these DNA crossovers appear to be critical to their recognition and processing into parental or recombinant products (Fig. 1).

We use nuclear magnetic resonance (NMR) spectroscopy to examine base-pairing, local structure, and conformational equilibria and time-resolved fluorescence resonance energy transfer (tr-FRET) to determine global folding arrangements and characterize conformational flexibility. Computation and molecular modeling are used to interpret and analyze the complementary NMR and tr-FRET data, and 3-dimensional structures in solution are generated by molecular dynamics calculations with NMR- and tr-FRET--derived restraints. Highlights for the past year include the development of new methods for region-specific labeling of DNA with NMR active isotopes, tr-FRET studies of an engineered Holliday junction analog with a novel backbone, and characterization of the interaction domain of the rpa2 gene product.

Our studies have shown that the key sequence-dependent structural property of a Holliday junction is the equilibrium distribution between the 2 crossover structural isomers (Fig. 2). We also discovered a previously unrecognized dependence of the structure and dynamics of Holliday junctions on the sequence adjacent to the branching point. Together, the results are providing evidence to distinguish between various structural models proposed for Holliday junctions and to determine how the sequence at the branching point modulates the global folding, local molecular conformation, and internal dynamics of these 4-arm DNA crossover structures.

In the past year, we developed tr-FRET methods to characterize mixed populations of DNA structural isomers, such as those observed for Holliday junctions and other branched DNA molecules. These methods are being used to determine the distributions of crossover isomers in a series of related model Holliday junctions as part of a systematic study of base-stacking preferences in the Holliday junction. In addition, we have undertaken tr-FRET studies of a novel Holliday junction analog that contains modified chemical linkages in the 2 crossover strands in an effort to understand the stereochemical features that govern the overall folding of the Holliday junction and the alignment of duplex stacking domains. New methods that use a DNA polymerase strategy to produce DNA oligomers have also been developed, and this protocol has been adapted to produce duplexes and Holliday junctions enriched in NMR active isotopes (¹³C, ¹⁵N). These labeled DNA molecules are being used to facilitate NMR analyses of Holliday junctions, damaged DNA, and complexes formed with recombination and repair enzymes. For example, complexes of uracil DNA glycosylase with uracil-containing DNA are being examined in an effort to decipher the orientation of the uracil during the various steps of processing by this enzyme.

Outstanding progress has also been made in determining the solution structure of a domain from the 32-kD subunit (rpa2 gene product) of replication protein A, which mediates interactions with numerous cellular targets, including uracil DNA glycosylase; p53; and the gene products of rad51, rad52, and xpa. The interactions between replication protein A and these proteins are essential for genetic recombination and for certain processes that repair damaged DNA. Biophysical characterization of these complexes with NMR spectroscopy is in progress.

PUBLICATIONS

Mer, G., Chazin, W.J. Enzymatic synthesis of region-specific isotope-labeled DNA for NMR analysis. J. Am. Chem. Soc. 120:607, 1998.

Structural Biology of Biomolecular Recognition, Binding, and Signal Transduction

W.J. Chazin, G. Bifulco,* J. Blankenship, D.L. Boger,** D.A. Case, J. Christodoulou, O. Crescenzi, P.A. Fagan, S. Forsén,*** L. Gomez-Paloma,* J. Harper,**** B. Huang,**** M.J. Hunter, R.R. Ketchem, S. Linse,*** L. Mäler, M.R. Nelson, K.C. Nicolaou,** M. Sastry, J. Schnell, J.A. Smith, E. Thulin,*** C. Weber

* University of Naples, Naples, Italy
** Department of Chemistry, TSRI
*** University of Lund, Lund, Sweden
**** Department of Cell Biology, TSRI

Our laboratory focuses on determining the structural basis of molecular recognition, binding, and triggering of cellular events critical to health and disease. We use nuclear magnetic resonance (NMR) spectroscopy in combination with a variety of biochemical, molecular biological, and computational methods to determine the solution structure and dynamics of proteins and oligonucleotides, free in solution and in complex with cellular targets or drugs. Highlights in the past year include solving a high-resolution structure of an important calcium sensor from the S100 subfamily (calcyclin), creating a new resource on the World Wide Web (the EF-Hand Calcium-Binding Protein Data Library), designing a novel calcium-binding protein (calbindomodulin), and solving the structure of duplex DNA alkylated by a novel duocarmycin antitumor agent.

CALCIUM SIGNAL TRANSDUCTION

Calcium and calcium-binding proteins (CaBPs) play a central role in intracellular signal transduction and are associated with a wide range of effects on health and disease. Our goal is to understand the molecular basis of the activation of CaBPs and the concomitant transduction of calcium signals. Ultimately, we hope to control binding properties and interactions with target proteins, thereby enabling the design of specific biological activities and therapeutic strategies relevant to calcium-mediated disease. We use NMR spectroscopy and computational methods to determine the 3-dimensional structures and internal dynamics of specific CaBPs in the presence and absence of calcium of cellular targets. Comparative structural analysis and protein engineering experiments are used to elucidate the fundamental principles that govern the calcium-induced activation of these proteins. These efforts are facilitated by the EF-Hand Calcium-Binding Protein Data Library, which integrates and organizes information on sequence, structure, and function.

We are determining the structure of several novel CaBPs. One of these is caltractin, an essential component of the microtubule organizing center in the centrosome that is required for accurate chromosomal segregation during the M stage (mitosis) of the cell cycle. Interest in caltractin is high because its phosphorylation in vivo apparently is cell-cycle dependent, a finding that suggests a role for this protein in coupling calcium and phosphorylation signaling pathways. In collaboration with B. Huang, Department of Cell Biology, we have developed high-level expression systems for the intact protein and for the 2 separate domains and have extensively characterized the biophysical properties of all 3 domains. Determination of the structure of caltractin in the presence and absence of calcium is in progress.

A second CaBP under study, in collaboration with J. Harper, Department of Cell Biology, is the plant calcium-dependent protein kinase. This unusual kinase apparently contains its own regulatory apparatus on the same protein chain: the kinase is homologous to mammalian calmodulin-dependent protein kinases but has an additional calmodulin-like domain at its C-terminus. Therefore, calcium-dependent activation of plant calcium-dependent protein kinase presumably mimics the calcium/calmodulin-dependent kinases, whereby binding by calmodulin of a flap helix on the kinase leads to exposure of the active site. Heteronuclear NMR is being used to analyze calcium activation and a purported calcium-independent binding to the junction region linking the kinase and the calmodulin domains.

We have placed a particular emphasis on the S100 subfamily of CaBPs. A growing body of evidence indicates that these proteins provide cell type--specific transduction of calcium signals. Members of the S100 subfamily have apparent cellular activities ranging from growth and proliferation to apoptosis and are implicated in a variety of disease states. The S100 protein calcyclin is preferentially expressed in the G₁ phase of the cell cycle, shows deregulated expression in association with cell transformation, and is found in high abundance in certain cancer cell lines. We determined the structure of native calcyclin in the apo state and found a novel organization of the calcium-binding domains (Fig. 1). We proposed that all other S100 proteins would have a similar structure and that their mode of signal transduction would be clearly distinct from that of classical calmodulin-like calcium sensors. This hypothesis was confirmed by solving the structure of the calcium-activated state (Fig. 1B).

Our research is being extended to the MRP8 and MRP14 proteins, which constitute a unique 2-chain S100 expressed by myeloid cells during inflammatory reactions. Inflammatory disorders such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis are associated with elevated levels of MRP8/MRP14. We recently showed a unique preferential affinity between the 2 subunits, and determination of the structure of the heterodimer complex is in progress.

One of the primary objectives of our research on CaBPs is to develop a fundamental understanding of the driving forces and molecular basis for signal transduction. For this purpose, we are using our EF-Hand Calcium-Binding Protein Data Library to analyze all known CaBP structures, and then, in collaboration with colleagues at the University of Lund, we are designing protein engineering experiments on the model system calbindin D_9k to test our hypotheses. Our current goal is to create a novel CaBP, calbindomodulin, by rationally redesigning calbindin D_9k to undergo a calmodulin-like opening upon binding calcium.

STRUCTURE-BASED DESIGN OF ANTITUMOR DRUGS

We are using 3-dimensional structures and molecular design to determine the structural basis of the antitumor activity of ligands that bind in the minor groove of duplex DNA. From this foundation, we are establishing a rational basis for the design of new antitumor drugs. Our current interests center on compounds that not only bind to but also react with the DNA substrate, because such agents often have exceptionally high biological potency.

One family of compounds under study in collaboration with K. Nicolaou, Department of Chemistry, is the calicheamicin DNA-scission agents. Recently, the structures of the head-to-head and head-to-tail dimers of the calicheamicin oligosaccharide domain have been obtained in complex with designed double-site DNA duplexes. These structures indicate the crucial role of oligosaccharides in recognition and binding of DNA and provide a basis for probing the inhibition of transcription by these compounds. Our work is one of the few examples of atomic resolution studies of biological recognition of carbohydrates.

A second family of compounds under investigation, in collaboration with D. Boger, Department of Chemistry, is the duocarmycin DNA alkylating agents. High-resolution structures of (+)-duocarmycin-SA and of a less reactive derivative lacking key functional groups, both bound to the same high-affinity DNA duplex, have been determined, (Fig. 2). Comparisons of the complexes formed with the parent compound and with the analog have provided critical new evidence confirming the hypothesis that the twist in the ligand induced by DNA binding governs the reactivity profiles and unique biological properties of these extremely potent alkylating agents.

PUBLICATIONS

Hunter, M.J., Chazin, W.J. High level expression and characterization of the S100 calcium-binding proteins MRP8 and MRP14. J. Biol. Chem. 273:12427, 1998.

Kördel, J., Pearlman, D.A., Chazin, W.J. Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D_9k. J. Biomol. NMR 10:231, 1997.

Kragelund, B.B., Jönnson, M., Bifulco, G., Chazin, W.J., Nilsson, H., Finn, B.E., Linse, S. Hydrophobic core substitutions in calbindin D_9k: Effects on Ca²⁺-binding and dissociation. Biochemistry, in press.

Nelson, M., Chazin, W.J. Calmodulin as a calcium sensor. In: Calmodulin and Signal Transduction. van Eldik, L.J., Watterson, D.M. (Eds.). Academic Press, San Diego, 1998, p. 17.

Nelson, M., Chazin, W.J. The EF-Hand Calcium-Binding Protein Data Library [on-line database]. Available at http://chazin.scripps.edu/capb-database.

Nelson, M., Chazin, W.J. An interaction-based analysis of calcium-induced conformational changes in Ca²⁺ sensor proteins. Protein Sci. 7:270, 1998.

Nelson, M., Weber, C., Chazin, W.J. Calcium-binding proteins. In: Encyclopedia of Molecular Biology. Creighton, T. (Ed.). Wiley, New York, in press.

Potts, B.C.M., Chazin, W.J. Chemical shift homology in proteins. J. Biomol. NMR 11:45, 1998.

Sastry, M., Ketchem, R.R., Crescenzi, O., Weber, C., Lubienski, M.J., Hidaka, H., Chazin, W.J. The three-dimensional structure of Ca²⁺-bound calcyclin: Implications for Ca²⁺-signal transduction by S100 proteins. Structure 6:223, 1998.

Skelton, N.J., Chazin, W.J. Solution structure determination of proteins using NMR spectroscopy. In: Peptide and Protein Analysis. Reid, R. (Ed.). Marcel Dekker, New York, in press.