The Skaggs Institute
for Chemical Biology

Scientific Report 2008

Macromolecular Master Keys for Genome integrity, Reactive Oxygen Control, and Pathogenesis

J.A. Tainer, A.S. Arvai, B.R. Chapados, L. Fan, C. Hitomi, K. Hitomi, J.J. Perry, M.E. Pique, D.S. Shin, J.L. Tubbs, R.S. Williams

To date, funds from the Skaggs Institute for Chemical Biology has supported the training of 15 graduate students and 33 postdoctoral fellows and contributions to 176 publications. In particular, Skaggs funding is used for medically relevant structure-function investigations of macromolecular master keys for genome integrity, reactive oxygen control, and pathogenesis. Skaggs funding also supports our synchrotron beam line, at the Advanced Light Source, University of California, Berkeley/Lawrence Berkeley National Laboratory. The synchrotron is used to characterize macromolecular complexes, conformations, and interactions in solution via small-angle x-ray scattering (SAXS) and at high resolution via macromolecular x-ray crystallography. We are furthermore developing and using new data interpretation tools for the detailed visualizations of protein complexes and modified proteins that undergo functionally important changes in shape and assembly. Working with P. Kuhn, Scripps Research, for example, we used our SAXS technologies to characterize solution structures that explain guanine nucleotide exchange mediated by the T-cell essential Vav1. We are working to develop SAXS for drug discovery so that the technology can be used to identify small molecules that bind and inhibit enzymes.

We are continuing research on pathogenic bacteria, including drug and vaccine design for the type IV pilin system. We have new structures for the fiber-forming type IV pilin protein, the assembly ATPase, and associated machinery. Type IV pilin proteins are critical bacterial virulence factors for cholera, pneumonia, gonorrhea, meningitis, and severe diarrhea. Our combined SAXS and macromolecular x-ray crystallography methods are providing a mechanistic understanding relevant to controlling these virulence factors.

In collaboration with M.N. Boddy, Scripps Research, we have identified the SUMO-targeted ubiquitin ligase family of proteins. These proteins provide communication between the sumoylation and ubiquitination pathways and act as master regulators of DNA damage responses. Thus, the proteins are of obvious value for the development of novel cancer drugs. We also defined new structure-function relationships for the multicomponent Smc5-Smc6 complex in genome stability.

Our work on macromolecules with key roles in pathways controlling reactive oxygen species and DNA damage responses is important for preserving the nervous system and controlling cancer. In our structure-based design projects, we collaborate with E.D. Getzoff, Skaggs Institute, to characterize inhibitors of nitric oxide synthase. In this collaborative work, we have discovered a new general method for designing inhibitors to specific isozymes of nitric oxide synthase that promise to reduce unwanted side effects and allow targeted interventions for arthritis, stroke, and cancer. Defining master keys to regulate reactive oxygen species such as superoxide and nitric oxide help provide an informed basis to avoid the oxidative death of neurons in neurodegenerative diseases and to use reactive oxygen species to kill cells involved in cancer and pathogenesis.

In other cancer related research, we characterized O⁶-alkylguanine-DNA alkyltransferase activity on x-linked DNA. This alkyltransferase is a target both for the prevention of cancer and for chemotherapy, because it repairs mutagenic lesions in DNA and limits the effectiveness of alkylating chemotherapies. We also used high-resolution crystal structures and mutational analyses to explain how endonuclease IV uses 3 metal ions to remove a damaged DNA site and then hold the product to avoid the release of toxic repair intermediates. We similarly characterized the role of active-site metal ions for flap endonuclease-1, which removes DNA flaps generated during both replication and repair.

In recent investigations on the nucleotide excision repair system, which repairs bulky lesions in DNA, we have focused on the helicase XPD. Our results define XPD helicase structures and activities to provide new insights into the cancer and aging phenotypes of XPD mutations (Fig. 1). Our findings also help explain the severe developmental problems associated with Cockayne syndrome, which includes defects in both DNA repair and transcription. Moreover, the effects of hijacking transcription factors and repair shielding associated with some XPD mutants may be the basis for the success of cisplatin as an anticancer agent. These results may therefore provide a mechanistic basis for the design if novel cancer agents that extend the therapeutic usefulness of cisplatin to other tumor types.

Fig. 1. Structural placement of disease-causing mutations in XPD helicase. Mapping the 3 classes of mutations onto the SaXPD structure reveals patterns associated with each disease defect. A, Stereopair mapping the distribution of disease-causing mutations on a XPD Cα trace. Disease-causing mutation sites (Cα colored sphere): red (XP), greenish yellow (XP/CS), and purple (TTD). Residue F136 is also shown (cyan). B, XPDcc fold and domain architecture (ribbons) with labeled disease-causing mutation sites as spheres colored as in A. C, XP mutations affect DNA- and ATP-binding regions. D, XP/CS mutations affect HD1-HD2 conformational changes. E, TTD mutations affect the overall framework stability. Reprinted from Fan, L., Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel, M., Roberts, V.A., Cooper, P.K., Tainer, J.A. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133:789, 2008, copyright 2008, with permission from Elsevier.

To understand the Mre11-Rad50-Nbs1 (MRN) complex that initiates repair of DNA double-strand breaks and homologous recombination, we are collaborating with P. Russell, Scripps Research. Our results help show how MRN mutations cause the Nijmegen breakage syndrome and ataxia telangiectasia–like disorder, diseases in humans that predispose individuals to cancer. Our new Mre11-DNA structures and mutants reveal key Mre11 roles in DNA end synapsis and nuclease processing (Fig. 2). We also identified Rad50 mutants that form meiotic DNA double-strand breaks and revealed an essential structural role for Rad50 in axial element and synaptonemal complex formation. In related research on homologous recombination, we used our new SAXS methods to define the domain structure interactions for the homologous recombination protein BARD1. We are now investigating the structural basis for homologous recombination; our results may have implications for improving cancer interventions by reprogramming cells for death vs repair in response to double-strand breaks.

Fig. 2. ATLD missense mutations W210C and N117S, which impair Nbs1 binding, cluster to a single Mre11 dimer surface opposite the DNA binding cleft.

Publications

Acharya, S.N., Many, A.M., Schroeder, A.P., Kennedy, F.M., Savytskyy, O.P., Grubb, J.T., Vincent, J.A., Friedle, E.A., Celerin, M., Maillet, D.S., Palmerini, H.J., Greischar, M.A., Moncalian, G., Williams, R.S., Tainer, J.A., Zolan, M.E. Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination. Genetics 180:1889, 2008.

Chrencik, J.E., Brooun, A., Zhang, H., Mathews, I.I., Hura, G.L., Foster, S., Perry, J.J., Streiff, M., Ramage, P., Widmer, H., Bokoch, G.M., Tainer, J.A., Weckbecker, G., Kuhn, P. Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1. J. Mol. Biol. 380:828, 2008.

Edwards, R.A., Lee, M.S., Tsutakawa, S.E., Williams, R.S., Tainer, JA., Glover, J.N.M. The BARD1 C-terminal domain structure and interactions with polyadenylation factor, CstF-50. Biochemistry 47:11446, 2008.

Fan, L., Fuss, J.O., Cheng, Q.J., Arvai, A.S., Hammel, M., Roberts, V.A., Cooper, P.K., Tainer, J.A. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133:789, 2008.

Fang, Q., Noronha, A.M., Murphy, S.P., Wilds, C.J., Tubbs, J.L., Tainer, J.A., Chowdhury, Q., Guengerich, F.P., Pegg, A.E. Repair of O⁶-G-alkyl-O⁶-G interstrand cross-links by human O⁶-alkylguanine-DNA alkyltransferase. Biochemistry 47:10892, 2008.

Garcin, E.D., Arvai, A.S., Rosenfeld, R.J., Kroeger, M.D., Crane, B.R., Andersson, G., Andrews, G., Hamley, P.J., Malinder, P.R., Nicholis, D.J., St-Gallay, S.A., Tinker, A.C., Gensmantel, N.P., Mete, A., Cheshire, D.R., Connolly, S., Stuhr, D.J., Alberg, A., Wallace, A.V., Tainer, J.A., Getzoff, E.D. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat. Chem. Biol. 4:700, 2008.

Garcin, E.D., Hosfield, D.J., Desai, S.A., Haas, B.J., Björas, M., Cunningham, R.P., Tainer, J.A. DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15:515, 2008.

Pebernard, S., Perry, J.J., Tainer, J.A., Boddy, M.N. Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability. Mol. Biol. Cell 19:4099, 2008.

Perry, J.J., Tainer, J.A. Structural biology of Cockayne syndrome proteins, their interactions and insights into DNA repair mechanisms. In: Molecular Mechanisms of Cockayne Syndrome. Ahmad, S.I. (Ed.). Landes Biosciences, Austin, TX, in press.

Perry, J.J.P., Tainer, J.A, Boddy, M.N. A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem. Sci. 33:201, 2008.

Syson, K., Tomlinson, C., Chapados, B.R., Sayers, J.R., Tainer, J.A., Williams, N.H., Grasby, J.A. Three metal ions participate in the reaction catalyzed by T5 flap endonuclease. J. Biol. Chem. 283:28741, 2008.

Williams, R.S., Moncalian, G., Williams, J.S., Yamada, Y., Limbo, O., Shin, D.S., Groocock, L.M., Cahill, D., Hitomi, C., Guenther, G., Moiani, D., Carney, J.P., Russell, P., Tainer, J.A. Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair. Cell 135:97, 2008.