Regulation of DNA Replication Licensing and Re-Replication by Cdt1

Abstract

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4^Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4^Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4^Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4^Cdt2. The proximity of PCNA-bound Cdt1 to CRL4^Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4^Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

Keywords: CRL4Cdt2; Cdt1; Cdt2; DNA re-replication; DNA repair synthesis; DNA replication; PCNA; Replication licensing; genome instability.

Figure 1

Both Cdt2 and its identified protein substrates contain the conserved PIP box motifs. (A) The consensus PIP box motifs in various CRL4^Cdt2 substrates. (B) The presence of a conserved PIP box-like motif in the Cdt2 proteins from different species.

Figure 2

A model for DNA synthesis and PCNA-dependent Cdt1 degradation by CRL4^CDT2. (A) During DNA replication or DNA damage-induced repair synthesis, Cdt1 and Cdt2 are recruited onto different subunits of the trimeric PCNA clamp encircling the replicating DNA strands. (B) The binding of Cdt1 and Cdt2 to the same trimeric PCNA clamp during DNA synthesis promotes the specific interaction between Cdt1 and CRL4^Cdt2 for the ubiquitin-dependent proteolysis of Cdt1 to prevent DNA re-replication and genome instability.