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.
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.
|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 Î· Mediates Its Interaction with the C-terminal Domain of Rev1. J Biol Chem 291:8735-44|
|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|
|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 23:724-33|