Mutations play a critical role in the etiology of many diseases, such as autism, schizophrenia, diabetes, and cancer. Most mutations arise during the replication of damaged DNA, a process called translesion synthesis (TLS). TLS is carried out by specialized TLS polymerases, which have evolved to accommodate DNA damage. Several fundamental, unanswered questions about TLS polymerases remain.
In Aim 1, we will address the following question: how are TLS polymerases recruited to stalled replication forks? To do this, we will use both ensemble and single-molecule binding assays and steady state kinetics. These studies will reveal the mechanisms of TLS polymerase recruitment to ubiquitin-modified PCNA (UbPCNA), a key component of stalled replication forks. These studies will also reveal the structural motifs required for these interactions.
In Aim 2, we will address the following question: how are TLS polymerases selected for recruitment to specific DNA lesions? To do this, we will use ensemble and single-molecule binding assay. These studies will reveal the mechanisms of TLS polymerase selection and the influence of the DNA lesion on this process.
In Aim 3, we will address the following question: how are TLS polymerases structurally organized at stalled replication fork? To do this, we will use X-ray crystallography, computational modeling and simulations, and a variety of experimental distance measurements. These studies will provide the first glimpse of the structural organization of TLS polymerases within these protein-DNA complexes and will reveal how the different TLS polymerases are coordinated during the multi-step process of TLS.

Public Health Relevance

Mutations, which play a critical role in causing many diseases, mainly arise during the replication of damaged DNA. Specialized enzymes called TLS polymerases are essential for this process. Understanding their detailed mechanisms, the objective of this proposal, will be important for developing novel strategies to eliminate mutations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM081433-06A1
Application #
8786802
Study Section
Special Emphasis Panel (ZRG1-GGG-Q (02))
Program Officer
Reddy, Michael K
Project Start
2008-07-01
Project End
2018-06-30
Budget Start
2014-08-15
Budget End
2015-06-30
Support Year
6
Fiscal Year
2014
Total Cost
$311,086
Indirect Cost
$91,675
Name
University of Iowa
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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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