Sequential replication-coupled destruction at G1/S ensures genome stability.
Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4(Cdt2), which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4(Cdt2) targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNA(DNA)) and recruits CRL4(Cdt2). Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNA(DNA). Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability.
Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Kate E
KE
Coleman
Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Gavin D
GD
Grant
Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; Curriculum in Bioinformatics and Computational Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Rachel A
RA
Haggerty
Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Kristen
K
Brantley
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA;
Etsuko
E
Shibata
Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Benjamin D
BD
Workman
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA;
Anindya
A
Dutta
Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
Dileep
D
Varma
Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; Curriculum in Bioinformatics and Computational Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Jeremy E
JE
Purvis
Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
Jeanette Gowen
JG
Cook
D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D016193:G1 Phase / Q000502:physiology*; D042822:Genomic Instability; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D016196:S Phase / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
C493956:CDKN1A protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C429530:DTL protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D044767:Ubiquitin-Protein Ligases
CDK; Cdt1; S phase; p21; replication stress; ubiquitin