Post-translational modifications of proliferating cell nuclear antigen (PCNA) are essential for maintaining genome stability. PCNA ubiquitylation facilitates translesion synthesis of damaged DNA templates, and PCNA SUMOylation prevents aberrant recombination. This proposal focuses on understanding the protein interactions regulated by post-translational modifications of PCNA and how these interactions modulate translesion synthesis and recombination. The proposed studies are possible because of a highly innovative technological development made by our group which allows for the production of large quantities of modified PCNA using a split/fusion strategy. Using this approach, we determined the X-ray crystal structures of both ubiquitin-modified PCNA (Ubi-PCNA) and SUMO-modified PCNA (SUMO-PCNA).
Aim 1 is to study the interaction and regulation of target proteins by Ubi-PCNA and SUMO-PCNA. Modified PCNA will be analyzed in single-molecule binding and reconstituted enzyme assays. These studies will test the tool belt model of PCNA action.
Aim 2 is to study the structure and dynamics of Ubi-PCNA and SUMO-PCNA. Structural studies will determine the dynamics of modified PCNA and test the hypothesis that dynamic protein-protein partner complexes are critical for regulation. Studies will also determine how post-translational modification of PCNA effects interactions with anti-recombinogenic helicases and modulates recombination.
Aim 3 is to study the regulation of DNA synthesis and recombination by Ubi-PCNA and SUMO-PCNA. The split/fusion methodology allows expression of constitutively modified PCNA in cells. This allows in vivo analysis of the effects of ubiquitylated and SUMOylated PCNA. These studies will provide a clear understanding of exactly how PCNA recruits proteins to replication forks, how the activities of different proteins bound to PCNA are regulated and coordinated, how the hand-off between different proteins occurs on PCNA during multi-step processes, and how post-translational modifications of PCNA have such different effects on these processes.

Public Health Relevance

Genomic instability drives carcinogenesis. Thus understanding the detailed mechanisms by which cells maintain genome stability is important for developing strategies to reduce genomic instability and carcinogenesis. The proposed work focuses on understanding how post-translational modifications of PCNA regulate error-prone translesion synthesis and aberrant recombination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM108027-02
Application #
8707499
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2013-08-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
2
Fiscal Year
2014
Total Cost
$332,651
Indirect Cost
$107,651
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
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, Elizabeth M; Spies, Maria; Washington, M Todd (2016) PCNA tool belts and polymerase bridges form during translesion synthesis. Nucleic Acids Res 44:8250-60
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 ? Mediates Its Interaction with the C-terminal Domain of Rev1. J Biol Chem 291:8735-44
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; Gildenberg, M S; Washington, M T (2016) The Many Roles of PCNA in Eukaryotic DNA Replication. Enzymes 39:231-54
Tsutakawa, Susan E; Yan, Chunli; Xu, Xiaojun et al. (2015) Structurally distinct ubiquitin- and sumo-modified PCNA: implications for their distinct roles in the DNA damage response. Structure 23:724-733