The long-term goal of our research is to understand the replication of damaged DNA in eukaryotes at the thermodynamic, kinetic, and structural levels. DNA damage in the template strand blocks replication by classical DNA polymerases. Consequently cells possess a variety of non-classical DNA polymerases that can replace the classical polymerase stalled at sites of DNA damage and can replicate through the damage. Recent kinetic studies and structural studies have provided substantial insights into how these non-classical polymerases differ from classical polymerases and how they are able to accommodate DNA damage. It remains unclear, however, how non-classical polymerases are recruited to sites of DNA damage, how stalled classical polymerases are displaced from sites of DNA damage, and how replication accessory factors promote nucleotide incorporation opposite DNA damage by non-classical polymerases. To address these issues, we propose studies with the following three specific aims: (1) to determine the effect of other protein factors on nucleotide incorporation opposite DNA damage by non-classical polymerases, (2) to determine the mechanism of non-classical polymerase recruitment during translesion synthesis, and (3) to determine the mechanism of classical polymerase displacement during translesion synthesis. These studies will provide a clear understanding of exactly how non-classical polymerases replace classical polymerases at sites of DNA damage and how other protein factors contribute to the replication of damaged DNA. Furthermore, these studies will contribute to our understanding of the origins of mutations and cancers and will provide insight into their prevention.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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University of Iowa
Schools of Medicine
Iowa City
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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
Kondratick, Christine M; Boehm, Elizabeth M; Dieckman, Lynne M et al. (2016) Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis. PLoS One 11:e0157023
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
Boehm, E M; Gildenberg, M S; Washington, M T (2016) The Many Roles of PCNA in Eukaryotic DNA Replication. Enzymes 39:231-54
Boehm, Elizabeth M; Powers, Kyle T; Kondratick, Christine M et al. (2016) The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1. J Biol Chem 291:8735-44
LuCore, Stephen D; Litman, Jacob M; Powers, Kyle T et al. (2015) Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures. Biophys J 109:816-26
Pryor, John M; Gakhar, Lokesh; Washington, M Todd (2013) Structure and functional analysis of the BRCT domain of translesion synthesis DNA polymerase Rev1. Biochemistry 52:254-63
Dieckman, Lynne M; Washington, M Todd (2013) PCNA trimer instability inhibits translesion synthesis by DNA polymerase η and by DNA polymerase δ. DNA Repair (Amst) 12:367-76
Dieckman, Lynne M; Boehm, Elizabeth M; Hingorani, Manju M et al. (2013) Distinct structural alterations in proliferating cell nuclear antigen block DNA mismatch repair. Biochemistry 52:5611-9
Dieckman, Lynne M; Freudenthal, Bret D; Washington, M Todd (2012) PCNA structure and function: insights from structures of PCNA complexes and post-translationally modified PCNA. Subcell Biochem 62:281-99

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