Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase.
DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup(ΔPIP)) and which cannot be regulated by CRL4(Cdt2). Dup(ΔPIP) is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup(ΔPIP) restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup(ΔPIP) accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.
Lee
Hyun O
HO
Curriculum in Genetics and Molecular Biology, Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.
Zacharek
Sima J
SJ
Xiong
Yue
Y
Duronio
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D048708:Cell Growth Processes / Q000502:physiology; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004330:Drosophila / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005260:Female; D064248:Geminin; D005786:Gene Expression Regulation; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D006080:Ovarian Follicle / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D016196:S Phase / Q000235:genetics* / Q000502:physiology; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; C411983:DUP protein, Drosophila; D029721:Drosophila Proteins; D064248:Geminin; D018809:Proliferating Cell Nuclear Antigen; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D004706:Endodeoxyribonucleases; C112206:DdbP1 protein, Drosophila; C568503:CRL4(Cdt2) protein, Drosophila
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