The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Macromolecular Machines as Master Keys for Genome Integrity, Control of Reactive Oxygen Species, and Pathogenesis

J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras, B.R. Chapados, L. Fan, C. Hitomi, K. Hitomi, I. Ivanov, J.J. Perry, M.E. Pique, C.D. Putnam, D.S. Shin, O. Sundheim, J.L. Tubbs, R.S. Williams, T.I. Wood, A. Yamagata

The Skaggs Institute funds our medically relevant projects on macromolecular machines. Skaggs also supports our synchrotron facilities at the Advanced Light Source, University of California, Berkeley/Lawrence Berkeley National Laboratory. We use small-angle x-ray scattering to characterize protein complexes, conformations, and interactions in solution and macromolecular x-ray crystallography for high-resolution studies. Macromolecular x-ray crystallography provides unparalleled structural detail critical for mechanistic analyses of macromolecules; small-angle x-ray scattering offers complementary information about folding, unfolding, aggregation, conformations, and assembly state in solution. Using these methods together, we can examine molecular interactions, flexibility, and conformational changes in solution that are relevant for accurate understanding, simulation, and prediction of mechanisms in structural biology and nanotechnology.

In our studies on microbial pathogens, we continue to characterize new drug targets for bacterial infectious diseases. We recently solved a new structure of a fiber-forming type IV pilin protein involved in bacterial pathogenesis and a target for vaccine development. Type IV pilins are bacterial virulence factors critical to controlling cholera, pneumonia, gonorrhea, meningitis, and severe diarrhea. We are also defining structures and conformations for the secretion ATPase superfamily. These results have implications for a universal secretion mechanism for pathogenesis.

In collaborations with E.D. Getzoff, Skaggs Institute, we have shown how green fluorescent protein (GFP) steers chemistry to favor fluorophore biosynthesis and disfavor alternative reactivity, and we have identified strategies for posttranslational modification and protein design. GFP creates its own fluorophore by promoting peptide backbone cyclization and amino acid oxidation on its own serine-tyrosine-glycine tripeptide sequence at positions 65–67. The posttranslational products identified from our GFP structures reveal mechanisms for carbon-bond cleavage, oxygen incorporation, and chromophore biosynthesis by GFP radical chemistry.

In collaboration with M.N. Boddy, Scripps Research, we identified the SUMO-targeted ubiquitin ligase protein family. These proteins establish a novel mode of communication between the sumoylation and ubiquitination posttranslational modification pathways; thus, we propose that the proteins are master regulators of DNA damage responses.

We are also investigating the structural chemistry of macromolecules with key roles in pathways that control reactive oxygen species and DNA damage responses; these pathways are essential for preserving the nervous system and controlling cancer. The structural studies may provide a basis for developing interventions to slow or prevent the progression of cancer and neurodegenerative diseases. For example, we defined a critical role for the zinc site of copper, zinc superoxide dismutase (SOD) in familial amyotrophic lateral sclerosis (FALS). More than 130 mutations in copper, zinc SOD result in the selective death of motor neurons. These SOD mutations occur in 25% of FALS patients. Despite their widespread distribution, mutations associated with FALS are positioned to cause structural and misfolding defects that decrease the affinity of SOD for zinc. Surprisingly, loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro.

To examine the importance of the zinc site in the structure of human SOD and the pathogenesis of FALS, we determined the crystal structure of a designed zinc-deficient human SOD. The results reveal the importance of SOD framework stability and suggest a role of zinc ion loss in the fatal neuropathologic changes associated with SOD mutations (Fig. 1).

Fig. 1. Loss of zinc affects the integrity of the SOD subunit fold and its dimer assembly, explaining why FALS mutations in humans are dominant.

For direct reversal of DNA base damage in humans, we are characterizing O⁶-alkylguanine-DNA alkyltransferase. This alkyltransferase is a crucial target for both prevention and chemotherapy because it repairs mutagenic lesions in DNA and it limits the effectiveness of alkylating chemotherapies. Our crystal structures of the human alkyltransferase provide an improved structural understanding of its interactions with biological substrates; these interactions are relevant to resistance to anticancer therapies.

For DNA repair by removal and replacement of the damaged area, we are discovering general principles along with detailed structural chemistry. Detection and repair of damaged DNA involve metastable complexes and distortion of both the repair proteins and DNA damage substrates; these distortions act as the driving force for repair chemistry and pathway coordination. Our structures include those involved in damage-specific excision initiated by structurally variable DNA glycosylases targeted to distinct base lesions, structure-specific nucleases, and proliferating cell nuclear antigen. Proliferating cell nuclear antigen forms a ring that provides binding sites for polymerase, flap endonuclease-1, and ligase during DNA replication and repair. Flap endonuclease-1 cleaves 5′-bifurcated nucleic acids at the junction formed between single- and double-stranded DNA. Our high-resolution structures provide detailed insights into how these proteins specifically recognize, remove, and repair DNA base damage without the release of toxic and mutagenic intermediates.

To understand repair of DNA double-strand breaks, we are characterizing the Mre11-Rad50-Nbs1 (MRN) complex, which is formed during the earliest response to such breaks. Mutations in MRN cause Nijmegen breakage syndrome and ataxia telangiectasia-like disorder, which are cancer predisposing. We are examining how the MRN complex orchestrates multiple requisite functional and conformational states in sensing and signaling in repair of double-strand breaks. The findings have general implications for ATP-binding cassette ATPases. In formation of the MRN complex, the Mre11 exonuclease directly binds Nbs1, DNA, and Rad50. Rad50, a structural maintenance of chromosome–related protein, uses its ATP-binding cassette ATPase, zinc hook, and coiled coils to bridge double-strand breaks and facilitate DNA end processing by Mre11 (Fig. 2).

Fig. 2. MRN is required for covalent modification and remodeling of chromatin at a double-strand break (DSB). The schematic is derived from our structural results and chromatin-remodeling events characterized in budding yeast. After induction of the break, MRN binds DNA ends and facilitates recruitment and activation of ATM-TEL1, which then phosphorylates nucleosomal histone H2A and expands outward over a large region of chromatin. The INO80 complex binds directly to gH2A to mediate histone eviction, an MRN-MRX–dependent process that facilitates timely DNA repair. The chromatin remodeler RSC also localizes to a double-strand break, aided by interaction with MRN, where it may mediate changes in chromatin structure that enhance MRN-dependent checkpoint signaling and DNA repair.

PUBLICATIONS

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. The case of the missing ring: radical cleavage of a carbon-carbon bond and implications for GFP chromophore biosynthesis. J. Am. Chem. Soc. 129:3118, 2007.

Hitomi, K., Iwai, S., Tainer, J.A. The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and repair. DNA Repair (Amst.) 6:410, 2007.

Ivanov, I., Chapados, B.R., McCammon, J.A., Tainer, J.A. Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications. Nucleic Acids Res. 34:6023, 2006.

Ivanov I., Tainer, J.A., McCammon, J.A. Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV. Proc. Natl. Acad. Sci. U. S. A. 104:1465, 2007.

Perry, J.J., Fan, L., Tainer, J.A. Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair. Neuroscience 145:1280, 2007.

Prudden, J., Pebernard, S., Raffa, G., Slavin, D.A., Perry. J.J., Tainer, J.A., McGowan, C.H., Boddy, M.N. SUMO-targeted ubiquitin ligases in genome stability. EMBO J. 26:4089, 2007.

Putnam, C.D., Hammel, M., Hura, G.L., Tainer, J.A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40:191, 2007.

Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R., Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. J. Mol. Biol. 373:877, 2007.

Tsutakawa, S.E., Hura, G.L., Frankel, K.A., Cooper, P.K., Tainer, J.A. Structural analysis of flexible proteins in solution by small angle x-ray scattering combined with crystallography. J. Struct. Biol. 158:214, 2007.

Tubbs, J.L., Pegg, A.E., Tainer, J.A. DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O⁶-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy. DNA Repair (Amst.) 6:1100, 2007.

Vijayakumar, S., Chapados, B.R., Schmidt, K.H., Kolodner, R.D., Tainer, J.A., Tomkinson, A.E. The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase. Nucleic Acids Res. 35:1624, 2007.

Williams, R., Sengerova, B., Osborne, S., Syson, K., Ault, S., Kilgour, A., Chapados, B.R., Tainer, J.A., Sayers, J.R., Grasby, J.A. Comparison of the catalytic parameters and reaction specificities of a phage and an archaeal flap endonuclease. J. Mol. Biol. 371:34, 2007.

Williams, R.S., Tainer, J.A. Learning our ABCs: Rad50 directs MRN repair functions via adenylate kinase activity from the conserved ATP binding cassette. Mol. Cell 25:789, 2007.

Williams, R.S., Williams, J.S., Tainer, J.A. Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem. Cell Biol. 85:509, 2007.

Yamagata, A., Tainer, J.A. Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J. 26:878, 2007.