CRL4(Cdt2) ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4(Cdt2) ubiquitin ligase. CRL4(Cdt2) targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4(Cdt2), PCNA, and a PCNA-binding degron motif on Pxd1. CRL4(Cdt2)-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4(Cdt2) ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.