Abstract
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 CRL4Cdt2. Here we provide evidence that CRL4Cdt2-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 CRL4Cdt2 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 CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage.
Highlights
Proliferating cell nuclear antigen (PCNA), a processivity factor for replicative DNA polymerases, acts as a docking molecular platform for many factors, and orchestrates several aspects of DNA metabolism such as DNA replication and repair [1]
On irradiation of cells with UV light, Pol Z is recruited to chromatin by interaction with PCNA and accumulates in discrete, microscopically visible foci that co-localize with sites of UV damage, visualized on expression of Pol Z fused to enhanced green fluorescent protein
We have provided evidence that activation of CRL4Cdt2 by DNA damage facilitates access of specific repair factors to PCNA, such as TLS Pol Z and Pol k, showing that CRL4Cdt2 plays an additional role in TLS DNA synthesis, independent of PCNA monoubiquitylation [62] involving efficient clearing of PIP degrons from PCNA on UV damage
Summary
Proliferating cell nuclear antigen (PCNA), a processivity factor for replicative DNA polymerases, acts as a docking molecular platform for many factors, and orchestrates several aspects of DNA metabolism such as DNA replication and repair [1]. Other PCNA partners, such as members of the Y-family of translesion synthesis DNA polymerases (TLS pols) that carry out DNA lesions bypass [7], require an ubiquitin-binding motif that tethers them to an ubiquitin group covalently attached to PCNA [8]. Monoubiquitylation of PCNA that occurs on DNA damage, increases the affinity of TLS pol Z for PCNA [9,10,11] and may constitute a mechanism to switch from replicative to TLS pols at stalled replication forks [12]. Pol Z is recruited at sites of ultraviolet (UV) damage on chromatin to bypass the major UV-induced DNA lesion, the thymine–thymine cyclobutane pyrimidine dimer photoproduct [13,14], and can be visualized by expression of eGFP-tagged Pol Z in cells [15].
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