A novel non-canonical PIP-box mediates PARG interaction with PCNA

Abstract

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG–PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites

“…Taken together, our results confirm that (i) the initial PARG recruitment is PARP1-dependent and faster relative to PCNA, and that (ii) PCNA promotes PARG stabilization at DNA damage sites [36,37], and reveal that (iii) in the absence of PARP1, PARG and PCNA are recruited with the same kinetic mechanism. Reproducing earlier findings validates our imaging method and analysis approach, while adding mechanistic details to the relative recruitment dynamics of PARG and PCNA in the presence and absence of PARP1.…”

“…While PARP1 depletion does not affect PCNA recruitment, as shown previously [34,36,53], it decreases PARG recruitment to DNA damage sites [36,37]. Using simultaneous dual-channel imaging we showed that in the absence of PARP1, PARG and PCNA are recruited with a comparable kinetic mechanism at a single cell level.…”

“…PARG is recruited initially through PARP1 and stabilised by PCNA [36,37]. PCNA and PARG form characteristic replication foci in S-phase cells, but exhibit a diffuse pattern in G1 and G2 phase of the cell cycle [36,37].…”

Targeting PCNA with Peptide Mimetics for Therapeutic Purposes

“…This indicates that the carboxylic acid group at position 6 is important for stabilising the binding motif . Similarly, position 6 Asp412 in a poly(ADP‐ribose) glycohydrolase (PARG) peptide does not contact PCNA; however, molecular dynamic simulations suggest that Asp412 P6 aids 3 10 ‐helix stabilisation . Pol δ p12 and Cdt1 also contain a Thr–Asp motif at PIP‐box position 5/6.…”

“…A wide variety of residues are incorporated at PIP‐box positions 2 and 3; however, a bias appears against negatively charged residues. Lys409 P3 in the PARG peptide plays a critical role in binding, with an Ala mutant showing no interaction with PCNA . The importance of this residue is attributed to both its positive charge, as a Lys409Arg P3 mutant recovered one‐third of the interaction with PCNA; as well as the hydrophobicity of the alkyl chain, as a Lys409Leu P3 mutant maintained PCNA binding affinity.…”