PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

• Published in: Nucleic acids research Vol. 42; no. 6; pp. 3692 - 3706
• Main Authors: Tsanov, Nikolay, Kermi, Chames, Coulombe, Philippe, Van der Laan, Siem, Hodroj, Dana, Maiorano, Domenico
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.2014
• Subjects: Animals; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Death; Cell Line; Cyclin-Dependent Kinase Inhibitor p21 - chemistry; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase - metabolism; Genetics; Genome Integrity, Repair and; Histone-Lysine N-Methyltransferase - chemistry; Humans; Life Sciences; Mice; NIH 3T3 Cells; Nuclear Proteins - metabolism; Protein Interaction Domains and Motifs; Proteolysis; Ubiquitin-Protein Ligases - metabolism; Ultraviolet Rays
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkt1400

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.