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)
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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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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
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 ýý and by DNA polymerase ýý. DNA Repair (Amst) 12:367-76
Freudenthal, Bret D; Brogie, John E; Gakhar, Lokesh et al. (2011) Crystal structure of SUMO-modified proliferating cell nuclear antigen. J Mol Biol 406:9-17
Pryor, John M; Washington, M Todd (2011) Pre-steady state kinetic studies show that an abasic site is a cognate lesion for the yeast Rev1 protein. DNA Repair (Amst) 10:1138-44
Tsutakawa, Susan E; Van Wynsberghe, Adam W; Freudenthal, Bret D et al. (2011) Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA. Proc Natl Acad Sci U S A 108:17672-7
Dieckman, Lynne M; Johnson, Robert E; Prakash, Satya et al. (2010) Pre-steady state kinetic studies of the fidelity of nucleotide incorporation by yeast DNA polymerase delta. Biochemistry 49:7344-50
Washington, M Todd; Carlson, Karissa D; Freudenthal, Bret D et al. (2010) Variations on a theme: eukaryotic Y-family DNA polymerases. Biochim Biophys Acta 1804:1113-23
Freudenthal, Bret D; Gakhar, Lokesh; Ramaswamy, S et al. (2010) Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange. Nat Struct Mol Biol 17:479-84
Freudenthal, Bret D; Gakhar, Lokesh; Ramaswamy, S et al. (2009) A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions. Acta Crystallogr D Biol Crystallogr 65:560-6

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