The Skaggs Institute
for Chemical Biology
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.
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
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
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
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
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
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.
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,
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.