Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis

Abstract

Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that prevents the stable incorporation of deoxyuridine monophosphate into DNA in the form of U/A basepairs. During S-phase, UNG2 remains associated with the replication fork through its interactions with two proteins, Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which are critical for DNA replication and repair. In this work, we used protein semisynthesis and fluorescence anisotropy assays to explore the interactions of UNG2 with PCNA and RPA and to determine the effects of two UNG2 phosphorylation sites (Thr and Tyr) located within its PCNA-interacting motif (PIP-box). In binding assays, we found that phosphorylation of Thr or Tyr on UNG2 can impede PCNA binding without affecting UNG2 catalytic activity or its RPA interaction. Our data also suggests that unmodified UNG2, PCNA, and RPA can form a ternary protein complex. We propose that the UNG2 N-terminus may serve as a flexible scaffold to tether PCNA and RPA at the replication fork, and that post-translational modifications on the UNG2 N-terminus disrupt formation of the PCNA-UNG2-RPA protein complex.

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA–dsDNA junctions

“…The procedures for the expression and purification of full-length hUNG2 ( 10 ) and the hUNG2 catalytic domain (amino acids 92–313) have been described previously ( 16 , 17 ). Heterotrimeric RPA was expressed and purified using the p11d-tRPA plasmid as described previously ( 10 , 18 , 19 ). The p11d-tRPA plasmid was a generous gift from Dr. Marc Wold.…”

“…To generate the isolated hUNG2 NTD (amino acids 1–91), a stop codon was inserted after residue 91 of hUNG2 in the pET21a vector described previously for the expression and purification of full-length hUNG2 ( 10 ). The encoded construct expressed in Escherichia coli contained an 8xHis-tag and the SUMO protein fused in-frame to the NTD of hUNG2.…”

“…The encoded construct expressed in Escherichia coli contained an 8xHis-tag and the SUMO protein fused in-frame to the NTD of hUNG2. Protein expression in BL21(DE3)pLysS cells, Ni 2+ column purification steps, and proteolytic removal of the 8xHis-SUMO tag was performed identically as for full-length hUNG2 ( 10 ). An additional column purification step was performed using a Superdex 75 10/300 GL column (GE Healthcare) with a mobile phase of 25 mM HEPES–NaOH (pH 7.4), 200 mM NaCl, 0.01% Triton X-100 and 1 mM TCEP.…”

“…Although the action of hUNG2 in class-switch recombination has not yet been associated with specific protein–protein interactions, the process requires active transcription and the formation of three-stranded DNA:RNA structures known as R-loops that recruit RPA ( 8 ). The importance of protein–protein interactions involving the NTD of hUNG2 is highlighted by the fact that these interactions are highly regulated by post-translational modifications ( 9 , 10 ), including phosphorylation events that promote binding to RPA ( 10 ).…”

Coordination of DNA Base Excision Repair by Protein-Protein Interactions

“…Based on these data, the acetylation status of TDG within tumor cells was proposed to impact the chemotherapy efficacy. Phosphorylation of the flexible N-terminus of DNA glycosylase UNG2 (at Thr6 or Tyr8 residues) shown to disrupt interaction with the PCNA factor, without affecting the UNG2 catalytic activity or its RPA interaction, has been proposed to regulate the formation of the ternary PCNA-UNG2-RPA protein complex [117].…”