Todd Washington, PhD

Portrait
Professor of Biochemistry
Professor of Radiation Oncology

Contact Information

Primary Office: 4-610 BSB
Iowa City, IA 52242
319-335-7518

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

Education/Training Program Affiliations

Department of Biochemistry PhD, Interdisciplinary Graduate Program in Molecular and Cellular Biology, Interdisciplinary Graduate Program in Translational Biomedicine, Medical Scientist Training Program

Center, Program and Institute Affiliations

Holden Comprehensive Cancer Center

Research Summary

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.

Publications

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. PMID: 26903512.

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. PMID: 27793278.

Washington, M. T. (2016). DNA Polymerase Fidelity: Beyond Right and Wrong. Structure (London, England : 1993), 24(11), 1855-1856. PMID: 27806256.

Boehm, E. M., Gildenberg, M. S. & Washington, M. T. (2016). The Many Roles of PCNA in Eukaryotic DNA Replication. The Enzymes, 39, 231-54. PMID: 27241932.

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. PMID: 27258147.

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. PMID: 27539869.

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. PMID: 27325737.

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. PMID: 25773143.

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. PMID: 26287633.

Pryor, J., Dieckman, L., Boehm, E. & Washington, T. (2014). Eukaryotic Y-Family Polymerases: A Biochemical and Structural Perspective. Springer (Eds.) pp. 5-10. Nucleic Acid Polymerases: Nucleic Acids and Molecular Biology.

Pryor, J. M., Gakhar, L. & Washington, M. T. (2013). Structure and functional analysis of the BRCT domain of translesion synthesis DNA polymerase Rev1. Biochemistry, 52(1), 254-63. PMID: 23240687.

Dieckman, L. M., Washington, M. T. (2013). PCNA trimer instability inhibits translesion synthesis by DNA polymerase η and by DNA polymerase δ. DNA repair, 12(5), 367-76. PMID: 23506842.

Dieckman, L. M., Boehm, E. M., Hingorani, M. M. & Washington, M. T. (2013). Distinct structural alterations in PCNA block DNA mismatch repair. Biochemistry, Epub ahead of print. PMID: 23869605.

Dieckman, L. M., Freudenthal, B. D. & Washington, M. T. (2012). PCNA Structure and Function: Insights from Structures of PCNA Complexes and Post-translationally Modified PCNA. Sub-cellular biochemistry, 62, 281-99. PMID: 22918591.

Washington, M. T., Freudenthal, B. F. & Kondratick, C. M. (2011). Crystal structures of ubiquitin-modified and SUMO-modified PCNA. Keystone Symposia on Molecular and Cellular Biology – DNA Replication and Recombination.

Tsutakawa, S. E., Van Wynsberghe, A. W., Freudenthal, B. D., Weinacht, C. P., Gakhar, L., Washington, M. T., Zhuang, Z., Tainer, J. A. & Ivanov, I. (2011). Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA. Proceedings of the National Academy of Sciences of the United States of America, 108(43), 17672-7. PMID: 22006297.

(2011). DNA Repair. McGraw-Hill Encyclopedia of Science and Technology.

(2011). Molecular Biology. McGraw-Hill Encyclopedia of Science and Technology.

Pryor, J. M., Washington, M. T. (2011). Pre-steady state kinetic studies show that an abasic site is a cognate lesion for the yeast Rev1 protein. DNA repair, 10(11), 1138-44. PMID: 21975119.

Freudenthal, B. D., Brogie, J. E., Gakhar, L., Kondratick, C. M. & Washington, M. T. (2011). Crystal structure of SUMO-modified proliferating cell nuclear antigen. Journal of molecular biology, 406(1), 9-17. PMID: 21167178.

(2011). PCR. McGraw-Hill Encyclopedia of Science and Technology.

Washington, M. T., Carlson, K. D., Freudenthal, B. D. & Pryor, J. M. (2010). Variations on a theme: eukaryotic Y-family DNA polymerases. Biochimica et biophysica acta, 1804(5), 1113-23. PMID: 19616647.

(2010). Cytochrome. McGraw-Hill Encyclopedia of Science and Technology.

Dieckman, L. M., Johnson, R. E., Prakash, S. & Washington, M. T. (2010). Pre-steady state kinetic studies of the fidelity of nucleotide incorporation by yeast DNA polymerase delta. Biochemistry, 49(34), 7344-50. PMID: 20666462.

Freudenthal, B. D., Gakhar, L., Ramaswamy, S. & Washington, M. T. (2010). Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange. Nature structural & molecular biology, 17(4), 479-84. PMID: 20305653.

(2010). Enzyme Inhibition. McGraw-Hill Encyclopedia of Science and Technology.

(2010). Steroid. McGraw-Hill Encyclopedia of Science and Technology.

Freudenthal, B. D., Ramaswamy, S. & Washington, M. T. (2009). Structural and Kinetic Studies of PCNA Mutant Proteins That Block Translesion Synthesis. American Society for Microbiology Conference on DNA Repair and Mutagenesis.

Freudenthal, B. D., Gakhar, L., Ramaswamy, S. & Washington, M. T. (2009). A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions. Acta crystallographica. Section D, Biological crystallography, 65(Pt 6), 560-6. PMID: 19465770.

Washington, M. T., Freudenthal, B. D., Ramaswamy, S. & Pryor, J. M. (2009). Role of Replication Accessory Factors in Translesion Synthesis. American Society for Microbiology Conference on DNA Repair and Mutagenesis.

Howell, C. A., Washington, M. T. (2008). Pre-Steady State Kinetic Studies of Protein-Template Directed Nucleotide Incorporation by the Yeast Rev1 Protein.” Keystone Symposia on Molecular and Cellular Biology – DNA Replication and Recombination.

Freudenthal, B. D., Ramaswamy, S. & Washington, M. T. (2008). Structure of a PCNA Mutant Protein That Blocks Translesion Synthesis.” Salk-Cal. Tech.-USC Meeting on DNA Replication and Genome Integrity.

Freudenthal, B. D., Ramaswamy, S. & Washington, M. T. (2008). Structure of a PCNA Mutant Protein That Blocks Translesion Synthesis.” Keystone Symposia on Molecular and Cellular Biology – DNA Replication and Recombination.

Howell, C. A., Kondratick, C. M. & Washington, M. T. (2008). Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta. Nucleic acids research, 36(5), 1731-40. PMID: 18263611.

Freudenthal, B. D., Ramaswamy, S., Hingorani, M. M. & Washington, M. T. (2008). Structure of a mutant form of proliferating cell nuclear antigen that blocks translesion DNA synthesis. Biochemistry, 47(50), 13354-61. PMID: 19053247.

Howell, C. A., Prakash, S. & Washington, M. T. (2007). Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein. Biochemistry, 46(46), 13451-9. PMID: 17960914.

(2006). Research Highlights: Mismatch Made in Heaven. Nat. Struct. Mol. Biol., 13, 956.

Carlson, K. D., Johnson, R. E., Prakash, L., Prakash, S. & Washington, M. T. (2006). Human DNA polymerase kappa forms nonproductive complexes with matched primer termini but not with mismatched primer termini. Proceedings of the National Academy of Sciences of the United States of America, 103(43), 15776-81. PMID: 17043239.

(2006). Research Highlights: Hitching a Ride. Nat. Chem. Biol., 6, 311.

Mason, A., Agrawal, N., Washington, M. T., Lesley, S. A. & Kohen, A. (2006). A lag-phase in the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mechanistic missing link. Chemical communications (Cambridge, England), 2006(16), 1781-3. PMID: 16609803.

Wolfle, W. T., Washington, M. T., Kool, E. T., Spratt, T. E., Helquist, S. A., Prakash, L. & Prakash, S. (2005). Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa. Molecular and cellular biology, 25(16), 7137-43. PMID: 16055723.

(2005). Glutathione. McGraw-Hill Encyclopedia of Science and Technology.

Carlson, K. D., Washington, M. T. (2005). Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase eta. Molecular and cellular biology, 25(6), 2169-76. PMID: 15743815.

(2005). Thyroid Hormone. McGraw-Hill Encyclopedia of Science and Technology.

(2005). Cytochrome. McGraw-Hill Encyclopedia of Science and Technology.

(2005). Dopamine. McGraw-Hill Encyclopedia of Science and Technology.

(2004). Steroids. McGraw-Hill Encyclopedia of Science and Technology.

(2004). Ribonuclease. McGraw-Hill Encyclopedia of Science and Technology.

Washington, M. T., Johnson, R. E., Prakash, L. & Prakash, S. (2004). Human DNA polymerase iota utilizes different nucleotide incorporation mechanisms dependent upon the template base. Molecular and cellular biology, 24(2), 936-43. PMID: 14701763.

(2004). Glucagon. McGraw-Hill Encyclopedia of Science and Technology.

Washington, M. T., Minko, I. G., Johnson, R. E., Haracska, L., Harris, T. M., Lloyd, R. S., Prakash, S. & Prakash, L. (2004). Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase zeta. Molecular and cellular biology, 24(16), 6900-6. PMID: 15282292.

Washington, M. T., Minko, I. G., Johnson, R. E., Wolfle, W. T., Harris, T. M., Lloyd, R. S., Prakash, S. & Prakash, L. (2004). Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases iota and kappa. Molecular and cellular biology, 24(13), 5687-93. PMID: 15199127.

Minko, I. G., Washington, M. T., Kanuri, M., Prakash, L., Prakash, S. & Lloyd, R. S. (2003). Translesion synthesis past acrolein-derived DNA adduct, gamma -hydroxypropanodeoxyguanosine, by yeast and human DNA polymerase eta. The Journal of biological chemistry, 278(2), 784-90. PMID: 12401796.

Washington, M. T., Wolfle, W. T., Spratt, T. E., Prakash, L. & Prakash, S. (2003). Yeast DNA polymerase eta makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate. Proceedings of the National Academy of Sciences of the United States of America, 100(9), 5113-8. PMID: 12692307.

Washington, M. T., Johnson, R. E., Prakash, L. & Prakash, S. (2003). The mechanism of nucleotide incorporation by human DNA polymerase eta differs from that of the yeast enzyme. Molecular and cellular biology, 23(22), 8316-22. PMID: 14585988.

Washington, M. T., Helquist, S. A., Kool, E. T., Prakash, L. & Prakash, S. (2003). Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta. Molecular and cellular biology, 23(14), 5107-12. PMID: 12832493.

Washington, M. T., Prakash, L. & Prakash, S. (2003). Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase eta. Proceedings of the National Academy of Sciences of the United States of America, 100(21), 12093-8. PMID: 14527996.

Wolfle, W. T., Washington, M. T., Prakash, L. & Prakash, S. (2003). Human DNA polymerase kappa uses template-primer misalignment as a novel means for extending mispaired termini and for generating single-base deletions. Genes & development, 17(17), 2191-9. PMID: 12952891.

Washington, M. T., Johnson, R. E., Prakash, L. & Prakash, S. (2002). Human DINB1-encoded DNA polymerase kappa is a promiscuous extender of mispaired primer termini. Proceedings of the National Academy of Sciences of the United States of America, 99(4), 1910-4. PMID: 11842189.

Haracska, L., Washington, M. T., Prakash, S. & Prakash, L. (2001). Inefficient bypass of an abasic site by DNA polymerase eta. The Journal of biological chemistry, 276(9), 6861-6. PMID: 11106652.

Minko, I. G., Washington, M. T., Prakash, L., Prakash, S. & Lloyd, R. S. (2001). Translesion DNA synthesis by yeast DNA polymerase eta on templates containing N2-guanine adducts of 1,3-butadiene metabolites. The Journal of biological chemistry, 276(4), 2517-22. PMID: 11062246.

Washington, M. T., Johnson, R. E., Prakash, S. & Prakash, L. (2001). Mismatch extension ability of yeast and human DNA polymerase eta. The Journal of biological chemistry, 276(3), 2263-6. PMID: 11054429.

Washington, M. T., Johnson, R. E., Prakash, L. & Prakash, S. (2001). Accuracy of lesion bypass by yeast and human DNA polymerase eta. Proceedings of the National Academy of Sciences of the United States of America, 98(15), 8355-60. PMID: 11459975.

Kondratick, C. M., Washington, M. T., Prakash, S. & Prakash, L. (2001). Acidic residues critical for the activity and biological function of yeast DNA polymerase eta. Molecular and cellular biology, 21(6), 2018-25. PMID: 11238937.

Washington, M. T., Prakash, L. & Prakash, S. (2001). Yeast DNA polymerase eta utilizes an induced-fit mechanism of nucleotide incorporation. Cell, 107(7), 917-27. PMID: 11779467.

Madril, A. C., Johnson, R. E., Washington, M. T., Prakash, L. & Prakash, S. (2001). Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7. The Journal of biological chemistry, 276(46), 42857-62. PMID: 11551952.

(2001). Silicon-Based Biology: The Road to the Virtual Cell.

Prakash, S., Johnson, R. E., Washington, M. T., Haracska, L., Kondratick, C. M. & Prakash, L. (2000). Role of yeast and human DNA polymerase Eta in Error-free replication of damaged DNA. Cold Spring Harbor Symp. Quant. Biol. LXV, 51-9.

(2000). Proteins and Protein Engineering: The Wet Side of Nanotechnology.

Washington, M. T., Johnson, R. E., Prakash, S. & Prakash, L. (2000). Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase eta. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 3094-9. PMID: 10725365.

Johnson, R. E., Washington, M. T., Prakash, S. & Prakash, L. (2000). Fidelity of human DNA polymerase eta. The Journal of biological chemistry, 275(11), 7447-50. PMID: 10713043.

Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S. & Prakash, L. (2000). Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature, 406(6799), 1015-9. PMID: 10984059.

(2000). Genes, Genomes, and Genome Projects: Biology in the New Century.

Washington, M. T., Johnson, R. E., Prakash, S. & Prakash, L. (1999). Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase eta. The Journal of biological chemistry, 274(52), 36835-8. PMID: 10601233.

Johnson, R. E., Washington, M. T., Prakash, L. & Prakash, S. (1999). Bridging the Gap: A family of novel DNA Polymerases for Mutagenic and error-free DNA damage bypass. Proceedings of the National Academy of Sciences of the United States of America, 96, 12224-26.

Patel, S. S., Hingorani, M. M., Washington, M. T., Moore, K. & Ahnert, P. (1998). Mechanisms of T7 DNA Helicase. (Vols. 74). pp. A334. Biophys Journal.

Washington, M. T., Patel, S. S. (1998). Improved DNA Unwinding Efficiency of Bacteriophage T7 Helicase Mutant Protein E348K. pp. A73. Biophys Journal.

Washington, M. T., Patel, S. S. (1998). Increased DNA unwinding efficiency of bacteriophage T7 DNA helicase mutant protein 4A'/E348K. The Journal of biological chemistry, 273(14), 7880-7. PMID: 9525882.

Hingorani, M. M., Washington, M. T., Moore, K. C. & Patel, S. S. (1997). The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase. Proceedings of the National Academy of Sciences of the United States of America, 94(10), 5012-17. PMID: 9144181.

Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W. & Patel, S. S. (1996). Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding. (Vols. 271). pp. 26825-34. The Journal of biological chemistry. PMID: 8900163.

Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S. & Studier, F. W. (1996). Selection, identification, and genetic analysis of random mutants in the clones primase/helicase gene of bacteriophage T7. (Vols. 271). pp. 26819-24. The Journal of Biological Chemistry.