Abstract

Ubiquitin-mediated proteolysis has diverse regulatory functions in eukaryotic cells (9759494). The role of ubiquitylation in regulating cell state by targeting proteins for proteasomal destruction is a major field of research. The ubiquitylation of a specific protein is performed by a ubiquitin-protein ligase (also known as E3) in an ATP-dependent manner. Ubiquitin is covalently bound to a ubiquitin-activating enzyme (E1), which transfers the ubiquitin to a ubiquitin-conjugating enzyme (E2). The E2 binds to the E3 ligase, which specifically recognises the target protein (12925736). There are two major types of E3 enzymes that ubiquitylate the substrate in different ways: HECT-type E3s first form an E3-ubiquitin thioester conjugate and then transfer the ubiquitin to the substrate. In contrast, RING-type E3s do not form this thioester bond but they coordinate zinc ions in a domain that binds the E2 (reviewed in 11395416 and 21828267). The cullin ring ligase 4 (CRL4) is a RING ubiquitin ligase containing the CUL4 as a scaffold protein, which is linked to the damage-specific DNA-binding protein 1 (DDB1), an adaptor, as well as to the RING domain protein Rbx1 that binds the E2. DDB1 recruits the DDB1/CUL4-associated factor (DCAF) representing the specific substrate-recognition subunit of CRL4^DCAF ubiquitin ligases.



The Proliferating Cell Nuclear Antigen (PCNA) is a homotrimer and forms a ring encircling double-stranded DNA. It slides along DNA as a clamp, which is required to ensure processivity of the DNA polymerase complex and of a broad range of other proteins involved in DNA metabolism. PCNA is loaded onto DNA during S phase or after DNA damage. This sliding clamp is highly conserved in Eukaroytes with homologues present in Bacteria, Archaea and some viruses (11056476). Proteins bind to PCNA via the PCNA interaction protein (PIP) box (LIG_PCNA). A subset of PIP box-carrying proteins contain a PIP box overlapping degron, termed PIP degron (LIG_CRL4_Cdt2_1; LIG_CRL4_Cdt2_2). This degron is recognised by the WD40 repeat domain of the Cdc10-dependent transcript 2 (Cdt2), the target recognition subunit of the CRL4^Cdt2 ubiquitin ligase. In order to ubiquitylate PIP degron-carrying proteins, the PIP box must first bind to chromatin-associated PCNA and subsequently, CRL4Cdt2 binds to the preformed complex. It has been observed that ubiquitylation of these PIP degron-carrying proteins by CRL4^Cdt2 occurs during S phase or after DNA damage.