Washington, Todd - University of Iowa - Department of Biochemistry

个人简介

Education BA, Philosophy, The Ohio State University BS, Biology, The Ohio State University PhD, Biochemistry, The Ohio State University Post Doctoral Fellow, University of Texas Medical Branch at Galveston

研究领域

Classical, replicative DNA polymerases synthesize DNA in a template-dependent fashion with remarkable efficiency and fidelity. They achieve rates as high as 1,000 nucleotide incorporations per second with error frequencies as low as one error per one million nucleotides incorporated. What these amazing enzymes cannot do, however, is replicate through DNA lesions that arise spontaneously or are formed upon attack by a plethora of DNA damaging agents including oxygen free radicals and radiation. Consequently, organisms have evolved specialized polymerases to replicate through lesions. DNA polymerase eta is one such specialized polymerase. Inactivation of DNA polymerase eta in yeast leads to an increase in the frequency of ultraviolet (UV) radiation-induced mutations. This indicates that the replication of UV- induced lesions by this polymerase is error-free ( i.e. , not mutagenic). In vitro , DNA polymerase eta has the unprecedented ability to accurately replicate through a thymine dimer, a common UV-induced lesion. Furthermore, defects in human DNA polymerase eta are responsible for the cancer prone genetic disorder, the variant form of xeroderma pigmentosum. DNA polymerase zeta is another specialized polymerase. Inactivation of DNA polymerase zeta in yeast leads to a dramatic decrease in the frequency of mutations induced by a wide range of DNA damaging agents. This indicates that the replication of numerous lesions by this polymerase is mutagenic. In vitro , DNA polymerase zeta has the remarkable ability to efficiently extend from primer- terminal mismatches containing template lesions. Thus, DNA polymerase zeta likely functions in the mutagenic replication of damaged DNA by extending from nucleotides inserted opposite lesions by other polymerases?often the classical, replicative polymerases themselves. Our long- term goal is to understand the mechanisms of DNA polymerases involved in both mutagenic and error-free replication of DNA damage at the thermodynamic, kinetic, and structural level. We use a variety of approaches including equilibrium binding techniques, transient state kinetic analyses (both rapid chemical quench flow and fluorescence-based stopped flow methods), and the characterization of mutant proteins generated by site-directed mutagenesis. We hope that this work will contribute to our understanding of the origins of mutations and cancers and perhaps gain new insights into their prevention.

近期论文

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Boehm, E. M., Powers, K. T., Kondratick, C. M., Spies, M., Houtman, J. C. & Washington, M. T. (2016). The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase ? Mediates Its Interaction with the C-terminal Domain of Rev1.. The Journal of biological chemistry, 291(16), 8735-44. DOI: 10.1074/jbc.M115.697938. Washington, M. T. (2016). DNA Polymerase Fidelity: Beyond Right and Wrong.. Structure (London, England : 1993), 24(11), 1855-1856. DOI: 10.1016/j.str.2016.10.003. Boehm, E. M., Gildenberg, M. S. & Washington, M. T. (2016). The Many Roles of PCNA in Eukaryotic DNA Replication.. The Enzymes, 39, 231-54. DOI: 10.1016/bs.enz.2016.03.003. Boehm, E. M., Subramanyam, S., Ghoneim, M., Washington, M. T. & Spies, M. (2016). Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.. Methods in enzymology, 581, 105-145. DOI: 10.1016/bs.mie.2016.08.019. Kondratick, C. M., Boehm, E. M., Dieckman, L. M., Powers, K. T., Sanchez, J. C., Mueting, S. R. & Washington, M. T. (2016). Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.. PloS one, 11(6), e0157023. DOI: 10.1371/journal.pone.0157023. Boehm, E. M., Washington, M. T. (2016). R.I.P. to the PIP: PCNA-binding motif no longer considered specific: PIP motifs and other related sequences are not distinct entities and can bind multiple proteins involved in genome maintenance.. BioEssays : news and reviews in molecular, cellular and developmental biology, 38(11), 1117-1122. DOI: 10.1002/bies.201600116. Boehm, E. M., Spies, M. & Washington, M. T. (2016). PCNA tool belts and polymerase bridges form during translesion synthesis.. Nucleic acids research, 44(17), 8250-60. DOI: 10.1093/nar/gkw563. LuCore, S. D., Litman, J. M., Powers, K. T., Gao, S., Lynn, A. M., Tollefson, W. T., Fenn, T. D., Washington, M. T. & Schnieders, M. J. (2015). Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures.. Biophysical journal, 109(4), 816-26. DOI: 10.1016/j.bpj.2015.06.062. Tsutakawa, S. E., Yan, C., Xu, X., Weinacht, C. P., Freudenthal, B. D., Yang, K., Zhuang, Z., Washington, M. T., Tainer, J. A. & Ivanov, I. (2015). Structurally distinct ubiquitin- and sumo-modified PCNA: implications for their distinct roles in the DNA damage response.. Structure (London, England : 1993), 23(4), 724-33. DOI: 10.1016/j.str.2015.02.008. Pryor, J., Dieckman, L., Boehm, E. & Washington, T. (2014). Eukaryotic Y-Family Polymerases: A Biochemical and Structural Perspective. pp. 5-10. Nucleic Acid Polymerases: Nucleic Acids and Molecular Biology.