CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation

Abstract

Structure-specific endonucleases are enzymes that process DNA intermediates generated in DNA replication, recombination, and repair. Proper regulation of these enzymes is critical for maintaining genome stability. Dna2 and XPF are two such enzymes present across eukaryotes, from yeasts to humans. Here, we show that in the fission yeastSchizosaccharomyces pombe, the activities of Dna2 and Rad16 (the equivalent of human XPF) are temporally controlled during the cell cycle by the CRL4(Cdt2) ubiquitin E3 ligase. In the S phase of the cell cycle, CRL4(Cdt2) promotes the degradation of Pxd1, which is an inhibitor of Dna2 and an activator of Rad16. Through targeting Pxd1 for degradation, CRL4(Cdt2) increases the activity of Dna2 in S phase and is important for the normal S-phase function of Dna2. Meanwhile, the degradation of Pxd1 reduces the activity of Rad16 in S phase, and curtails Rad16-dependent single-strand annealing, a mutagenic DNA repair pathway. Our findings uncover a new mechanism regulating two important endonucleases during the cell cycle, and reveal a new way of coordinating endonucleases to safeguard genome stability. 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 yeastSchizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 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 CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-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 CRL4Cdt2 yubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.