Faculty / Research
Miguel Garcia-Diaz, PhDAssociate Professor
Director, Molecular and Cellular Pharmacology Graduate Program
PhD, Universidad Autonoma, Madrid (Spain)
Postdoctoral, National Institute of Environmental Health Sciences. Laboratory of Molecular Genetics and Laboratory of Structural Biology
Mechanisms of mitochondrial gene expression/Mechanisms of DNA synthesis and repair
Mitochondrial gene expression and mitochondrial disease
Mitochondria contain a small (16.5 kb) double-stranded circular genome that in mammals encodes 13 polypeptides. Expression of this genome depends on a molecular genetic system that is distinct from the nuclear gene expression machinery. Defects in mitochondrial gene expression, often resulting from the inheritance of genetic mutations, result in mitochondrial dysfunction and different human diseases. Our work is trying to understand several essential processes for gene expression in mitochondria, and how deficiencies in these processes are related to pathogenesis.
Mechanisms of mitochondrial transcription
Mitochondrial transcription is a carefully regulated process that is crucial to generate the tRNAs, rRNAs and mRNAs necessary for mitochondrial protein production, but is also essential to initiate replication of the mitochondrial genome. How this process is specifically organized and regulated is still a matter of debate, but it involves the RNA polymerase, at least two transcription factors and no more than two or three promoters that generate polycistronic transcripts that are subsequently processed. Understanding the mechanisms by which the cell controls differential initiation at these promoters is key to understand how the transcription process is regulated. We are studying the process of transcription initiation taking advantage of a robust reconstituted in vitro system and a combination of single-particle electron microscopy, small-angle x-ray scattering and biochemical and biophysical measurements.
Transcription termination and MTERF proteins
Another aspect of mitochondrial transcription that we are investigating is the mechanism of transcription termination at a specific site downstream of the rRNA genes. This termination event is dependent on a protein called MTERF1, for Mitochondrial TERmination Factor 1, and it was postulated to be important to determine the ratio of mitochondrial rRNAs to mRNAs, although this is still a subject of debate. We have demonstrated that MTERF1 belongs to a class of modular proteins characterized by an all-alpha-helical fold. We also found that MTERF1 utilizes an extremely unusual DNA binding mechanism that involves active distortion of the DNA molecule and demonstrated that several disease-associated mitochondrial DNA mutations can interfere with this binding mode, severely impairing the termination process. This suggests that their effect on MTERF1 function might be related to the pathogenic mechanism.
Mitochondrial rRNA modifications
We are studying the process of rRNA modification in mitochondria. Although the functional significance of these modifications is unclear, they are essential for mitochondrial function. We have studied the roles in this process of MTERF4, an MTERF family member, and TFB1M, a methyltransferase responsible for adenine dimethylation in a conserved stem-loop near the 3’-end of the 12S rRNA.
DNA synthesis and DNA repair
The nuclear replication process in humans is extremely complex and only partially understood. The accuracy of this process is essential to avoid deleterious mutagenesis. The proteins mainly responsible for this accuracy are DNA polymerases, the enzymes responsible for DNA synthesis. There are at least 15 different DNA polymerases in human cells, and except for a few their functions are largely unknown. Structural analysis of DNA polymerases has made a significant contribution to our understanding of DNA polymerase fidelity and substrate specificity, and this information is precious to understand their biological function.
We are particularly interested in DNA polymerases responsible for smaller patches of DNA synthesis that mainly take place during DNA repair. We are studying how these enzymes contribute to genome stability, in particular in response to DNA damage, and how their function can be affected by different environmental exposures.
Mejia, E. Burak, M., Alonso, A., Larraga, V., Kunkel, T.A., Bebenek, K., Garcia-Diaz, M. (2014) Structures of the Leishmania infantum polymerase beta. DNA Repair (in press).
Venkataraman, K., Guja, K.E., Garcia-Diaz, M., Karzai, A.W. (2014) Non-stop mRNA decay: a special attribute of trans-translation mediated ribosome rescue. Front. Microbiol. 5:93.
Pan, P., Knudson, S.E., Bommineni G.R., Li, H.J., Lai, C.T., Liu, N., Garcia-Diaz, M., Simmerling, C., Patil, S.S., Slayden, R.A., Tonge, P.J. (2014) Time-dependent diaryl ether inhibitors of InhA:structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy. ChemMedChem 9, 776-791.
Li, H.J., Lai, C.T., Pan, P., Yu, W., Liu, N., Bommineni, G.R., Garcia-Diaz, M., Simmerling, C., Tonge, P.J. (2014) A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA. ACS Chem. Biol. 9, 986-993.
Yakubovskaya, E., Guja, K.E., Eng, E.T., Choi, W.S., Mejia, E., Beglov, D., Lukin, M., Kozakov, D., Garcia-Diaz, M. (2014) Organization of the human mitochondrial transcription initiation complex. Nucleic Acids Res. 42, 4100-4012.
Garrido, P., Mejia, E., Garcia-Diaz, M., Blanco, L., Picher, A.J. (2014) The active site of TthPolX is adapted to prevent 8-oxo-dGTP misincorporation. Nucleic Acids Res. 42, 534-543.
Jacewicz, A., Trzemecka., A., Plochicka, D., Bebenek, A., Garcia-Diaz., M. (2013) A remote palm domain residue of RB69 DNA polymerase determines the
conformation of the active site and is critical for enzyme activity and fidelity. PLOS One 8:e76700.
Guja, K., Venkataraman, K., Yakubovskaya, E., Karzai, A.W., Garcia-Diaz, M. (2013) Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1. Nucleic Acids Res. 41, 7947-7959.
Yakubovskaya, E., Guja, K., Mejia, E., Castano, S., Hambardjieva, E., Choi, W., Garcia-Diaz, M. (2012) Structure of the essential MTERF4:NSUN4 protein complex reveals how an MTERF protein collaborates to facilitate rRNA modification. Structure 20, 1940-1947.
Bruni, F., Manzari, C., Filice, M., Loguericio Polosa, P., Colella, M., Carmone, C., Hambardjieva, E., Garcia-Diaz., M., Cantatore, P., Roberti, M. (2012) D-MTERF5 is a novel factor modulating transcription in D. melanogaster mitochondria. Mitochondrion 12, 492-499.
Guja, K., Garcia-Diaz, M. (2012) Hitting the brakes: termination of mitochondrial transcription. BBA 1819, 939-947.
Li, H., Li, X., Liu, N., Zhang, H., Truglio, J., Mishra, S. Kisker, C., Garcia-Diaz, M., Tonge, P. (2011) Mechanism of the Intramolecular Claisen Condensation Reaction Catalyzed by MenB, a Crotonase Superfamily Member. Biochemistry 50, 9532-9544.
Babychuk, E., Vandepoele, K., Wissing, J., Garcia-Diaz, M., De Rycke, R., Akbari, H., Joubes, J., Beeckman, T., Jansch, L., Frentzen, M., Van Montagu, M.C., Kushnir, S. (2011) Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family. Proc. Natl. Acad. Sci. USA 108, 6674-6679.
Byrnes, J., Garcia-Diaz, M. (2011) Mitochondrial transcription: how does it end? Transcription 2, 32-36
Yakubovskaya, E., Mejia, E., Byrnes, J., Hambardjieva, E., Garcia-Diaz, M. (2010) Helix unwinding and base flipping enable human MTERF1 to terminate mitochondrial transcription. Cell 141, 982-993.
Bebenek, K., Garcia-Diaz, M., Zhou, R.Z., Povirk, L.F., Kunkel, T.A. (2010) Loop 1 modulates the fidelity of DNA polymerase lambda. Nucleic Acids Res 38, 5419-5431.
Garcia-Diaz, M., Murray, M.S., Kunkel, T.A., Chou, K.M. (2010) Interaction between DNA polymerase lambda and anticancer nucleoside analogs. J. Biol. Chem. 285, 16874-16879.
Nassar, N, Singh, K., Garcia-Diaz, M. (2010) Structure of the dominant negative S17N mutant of Ras. Biochemistry 49, 1970-1974.
Scotto-Lavino, E., Garcia-Diaz, M., Du, G., Frohman, M.A. (2010) Basis for the isoform-specific interaction of myosin phosphatase subunits protein phosphatase 1c beta and myosin phosphatase targeting subunit 1. J. Biol. Chem. 285, 6419-6424.
Terrados G, Capp JP, Canitrot Y, Garcia-Diaz M, Bebenek K, Kirchhoff T, Villanueva A, Boudsocq F, Bergoglio V, Cazaux C, Kunkel TA, Hoffmann JS, Blanco L. (2009) Characterization of a natural mutator variant of human DNA polymerase lambda which promotes chromosomal instability by compromising NHEJ. PloS One 4, e7290.
Garcia-Diaz, M, Bebenek, K, Larrea, A.A., Havener, J.M., Perera, L., Krahn, JM, Pedersen, L.C., Ramsden, D.A., Kunkel, TA (2008) Template strand scrunching during DNA gap repair synthesis. Nat. Struct. Mol. Biol. 9, 967-972.
Cisneros, GA, Perera, L, Garcia-Diaz, M, Bebenek, K, Kunkel, TA, Pedersen LG (2008) Catalytic mechanism of humjan DNA polymerase lambda with Mg2+ and Mn2+ from ab initio QM/MM studies. DNA Repair 7, 1824-1834.
Zhou, RZ, Blanco, L, Garcia-Diaz, M, Bebenek, K, Kunkel, TA, Povirk, LF (2008) Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap-filling of partially complementary double-strand break ends by DNA polymerase l in human nuclear extracts. Nucleic Acids Res. 36, 2895-2905.
Bebenek, K, Garcia-Diaz, M, Foley, MC, Pedersen, LC, Schlick, T, Kunekl, TA (2008) Substrate-induced DNA strand misalignment during catalytic cycling by DNA polymerase lambda. EMBO Rep. 9, 459-464.
Moon, AF, Garcia-Diaz, M, Batra, VK, Beard, WA, Bebenek, K, Kunkel, TA, Wilson, SH, Pedersen, LC. (2007) The X family portrait: Structural insights into biological functions of X family polymerases. DNA Repair 6, 1709-1725.
Garcia-Diaz, M, Bebenek, K, Pedersen, L, Kunkel, T. (2007) Role of the catalytic metals during polymerization by DNA polymerase lambda. DNA Repair 6, 1333-1340.
Garcia-Diaz, M, Bebenek, K. (2007) Multiple functions of DNA polymerases. Crit. Rev. Plant Sci. 26, 105-122.
Moon A.F., Garcia-Diaz, M., Bebenek, K., Davis, B.J., Zhong, X., Ramsden, D.A., Kunkel, T.A., Pedersen, L. (2007) Structural insight into the substrate specificity of DNA Polymerase mu. Nat. Struct. Mol. Biol. 14, 45-53.
Garcia-Diaz, M., Kunkel, T.A. (2006) Mechanism of a genetic glissando:structural biology of indel mutations. Trends in Biochem. Sci. 31, 206-214.
Garcia-Diaz, M., Benenek, K., Krahn, J.M., Pedersen, L.C. and Kunkel, T.A. (2006) Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase. Cell 124, 331-342.
Nick McElhinny, S.A., Havener, J.M., Garcia-Diaz, M., Juarez, R., Bebenek, K., Kee, B.L., Blanco, L., Kunkel, T.A. and Ramsden, D.A. (2005) A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous en joining. Mol. Cell 19, 357-366.
Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Kunkel, T.A., Pedersen, L.C. (2005) A closed conformation for the Pol lambda catalytic cycle. Nat. Struct. Mol. Biol. 12, 97-98.
Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Blanco, L., Kunkel, T.A., Pedersen, L.C. (2004) A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology. Mol. Cell 13, 561-572.
Bebenek, K., Garcia-Diaz, M., Blanco, L., Kunkel. T.A. (2003) The frameshift infidelity of human DNA polymerase lambda. J. Biol. Chem. 278, 34685-34690.
Garcia-Diaz, M., Bebenek, K., Sabariegos, R., Dominguez, O., Rodriguez, J., Kirchhoff, T., Garcia-Palomero, E., Picher, A.J., Juarez, R., Ruiz, J.F., Kunkel, T.A., Blanco, L. (2002) DNA polymerase lambda, a novel DNA repair enzyme in human cells. J. Biol. Chem. 277, (13184-13191).
Garcia-Diaz, M., Bebenek, K., Kunkel, T., Blanco, L. (2001) Identification of an intrinsic dRP lyase activity in human DNA polymerase lambda: a possible role in base excision repair. J Biol Chem 276, (34659-34663).
García-Díaz, M., Domínguez, O., López-Fernández, L., Laín de Lera, T., Saníger, M.L., Ruiz, J.F., Párraga, M., García, M.J., Kirchhoff, T., del Mazo, J., Bernad, A., Blanco, L. (2000) DNA polymerase lambda (Pol l), a novel eukaryotic DNA polymerase. J Mol Biol, 301 (851-867).
Dominguez, O., Ruiz, J.F., Lain, T., Garcia-Diaz, M., Gonzalez, M., Kirchhoff, T., Martinez-A., C., Bernad, A., Blanco, L. (2000) DNA polymerase mu (Pol m), homologous to TdT, could act as a DNA mutator in eukaryotic cells. EMBO J, 19 (1731-1742).