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).
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.