Detection of Multi‐Protein Complexes Containing PCNA Using Fluorescence Anisotropy and Hormetic Modeling

Abstract

Proliferating Cell Nuclear Antigen (PCNA) is a homotrimeric protein involved in DNA replication and repair. It contains three equivalent binding sites that are known to interact with dozens of replication and repair proteins. In theory, PCNA could bind multiple proteins at the same time, but steric hindrance prevents this for some protein combinations. Here, we developed an approach to detect the formation of multi‐protein complexes containing PCNA and base excision repair proteins using fluorescence anisotropy and hormetic modeling. Initially, we measured binding of fluorescent‐labeled pogo‐ligase peptide (PL) to PCNA and determined a Kdof 118 nM. This assay measured an increase in the fluorescence anisotropy of 50 nM PL upon PCNA binding because the fluorescent peptide becomes part of a larger macromolecular complex that slows its movement in solution (anisotropy min/max: 0.043/0.121). Next, we competed 50 nM PL from 0.25 μM PCNA using uracil DNA glycosylase (UNG2), which reduced the anisotropy from 0.086 to 0.057, and we determined an IC50 of 4 μM with a conventional sigmoidal curve. In contrast, displacement of PL from 3 μM PCNA using UNG2 resulted in a hormetic dose response that was fit using the Brain‐Cousens equation for hormesis. In these experiments, the anisotropy of 50 nM PL increased from 0.095 in the presence of 3 μM PCNA to 0.127 with the addition of 5 μM UNG2, and the anisotropy then reduced to 0.086 with 50 μM UNG2. The hormetic response occurred because a ternary PCNA‐PL‐UNG2 complex formed at UNG2 concentrations that were sufficient to bind open PCNA sites, but were insufficient to displace PL from PCNA, and the anisotropy of PL bound to the ternary complex was higher than its anisotropy bound to PCNA alone. Additional values derived from the Brain‐Cousens curve included a significant hormesis parameter f (0.024), a maximum stimulatory response of 134% of control at a dose of 4 μM UNG2, and a limited dose for stimulation at 31 μM UNG2. We also report additional datasets where UNG2 displaced 50 nM PL from other PCNA concentrations to train our hormetic modeling and optimize assay signal/noise. Continuing experiments using fluorescent‐labeled UNG2 and fluorescent‐labeled DNA polymerase β (POLB), in addition to DNA Ligase 1 (LIG1), will explore the simultaneous and/or sequential interactions of base excision repair proteins with PCNA.