Mutations play a fundamental role in the etiology of many diseases including cancers, diabetes, epilepsy, schizophrenia, and others. Many of these mutations result from translesion synthesis, a pathway the cell uses to bypass DNA damage during DNA replication. In translesion synthesis, the high-fidelity classical DNA polymerase (i.e., the one involved in normal DNA replication) encounters DNA damage and stalls. Low-fidelity non-classical polymerases are recruited to the stalled replication fork where they carry out replication of the damaged DNA. Each non-classical polymerase is specialized for incorporating nucleotides opposite a few types of DNA damage. When the proper non-classical polymerase is used, damage bypass is not mutagenic. When an improper one is used, it is mutagenic. The overall goal of the proposed research is to understand the factors that affect the accuracy and efficiency of translesion synthesis. We are particularly interested in examining how non-classical polymerases are selected and regulated during translesion synthesis. Little is known about non-classical polymerase selection and regulation, because these polymerases function within dynamic, multi-protein complexes (bypass complexes) that are difficult to study. We are using an innovative and novel combination of biochemical, biophysical, and structural approaches that will allow us to overcome these challenges.
In Aim 1, we will examine the architecture of bypass complexes using single-molecule total internal reflection fluorescence (TIRF) experiments.
In Aim 2, we will examine the regulation of bypass complexes using steady state and pre-steady state kinetics studies.
In Aim 3, we will examine the structure of bypass complexes using X-ray crystallography, small-angle X-ray scattering (SAXS) and Brownian dynamics (BD) simulations.

Public Health Relevance

Mutations, which play a fundamental role in causing many diseases, mainly arise during the replication of damaged DNA. A class of enzyme called non-classical polymerases are essential for this process. Understanding how non-classical polymerases are selected to replicate the damaged DNA and how they are regulated ? the objectives of this proposal ? will be important for developing novel strategies to eliminate disease-causing mutations.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Reddy, Michael K
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University of Iowa
Schools of Medicine
Iowa City
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Powers, Kyle T; Elcock, Adrian H; Washington, M Todd (2018) The C-terminal region of translesion synthesis DNA polymerase ? is partially unstructured and has high conformational flexibility. Nucleic Acids Res 46:2107-2120
Powers, Kyle T; Washington, M Todd (2018) Eukaryotic translesion synthesis: Choosing the right tool for the job. DNA Repair (Amst) :
Kondratick, Christine M; Litman, Jacob M; Shaffer, Kurt V et al. (2018) Crystal structures of PCNA mutant proteins defective in gene silencing suggest a novel interaction site on the front face of the PCNA ring. PLoS One 13:e0193333
Powers, Kyle T; Lavering, Emily D; Washington, M Todd (2018) Conformational Flexibility of Ubiquitin-Modified and SUMO-Modified PCNA Shown by Full-Ensemble Hybrid Methods. J Mol Biol 430:5294-5303
Liu, Jie; Ede, Christopher; Wright, William D et al. (2017) Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase ?-extending D-loops. Elife 6:
Zhao, Linlin; Washington, M Todd (2017) Translesion Synthesis: Insights into the Selection and Switching of DNA Polymerases. Genes (Basel) 8:
Powers, Kyle T; Washington, M Todd (2017) Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases. Methods Enzymol 592:329-356
Boehm, Elizabeth M; Washington, M Todd (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 38:1117-1122
Washington, M Todd (2016) DNA Polymerase Fidelity: Beyond Right and Wrong. Structure 24:1855-1856
Boehm, E M; Subramanyam, S; Ghoneim, M et al. (2016) Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy. Methods Enzymol 581:105-145

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