Cdt1 and Cdc6 are destabilized by rereplication-induced DNA damage.
The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that metazoan origins initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion or overexpression of Cdt1 or Cdc6 in human cells causes rereplication, a form of endogenous DNA damage. Rereplication induced by these manipulations is however uneven and incomplete, suggesting that one or more mechanisms restrain rereplication once it begins. We find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. We further show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4(DDB1) ubiquitin ligase, and Cdt1 can induce its own degradation when overproduced. Cdc6 degradation in geminin-depleted cells requires Huwe1, the ubiquitin ligase that regulates Cdc6 after DNA damage. Moreover, perturbations that specifically disrupt Cdt1 and Cdc6 degradation in response to DNA damage exacerbate rereplication when combined with geminin depletion, and this enhanced rereplication occurs in both human cells and in Drosophila melanogaster cells. We conclude that rereplication-associated DNA damage triggers Cdt1 and Cdc6 ubiquitination and destruction, and propose that this pathway represents an evolutionarily conserved mechanism that minimizes the extent of rereplication.
Hall
Jonathan R
JR
Department of Biochemistry and Biophysics, Chapel Hill, North Carolina 27599-7260.
Lee
Hyun O
HO
Department of Curriculum in Genetics and Molecular Biology, Chapel Hill, North Carolina 27599-7260.
Bunker
Brandon D
BD
Department of Biochemistry and Biophysics, Chapel Hill, North Carolina 27599-7260.
Dorn
Elizabeth S
ES
Department of Biochemistry and Biophysics, Chapel Hill, North Carolina 27599-7260.
Rogers
Greg C
GC
Department of Biology, Chapel Hill, North Carolina 27599-7260.
Duronio
Robert J
RJ
Department of Curriculum in Genetics and Molecular Biology, Chapel Hill, North Carolina 27599-7260; Department of Biology, Chapel Hill, North Carolina 27599-7260; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260.
Cook
Jeanette Gowen
JG
Department of Biochemistry and Biophysics, Chapel Hill, North Carolina 27599-7260; Department of Curriculum in Genetics and Molecular Biology, Chapel Hill, North Carolina 27599-7260; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260. Electronic address: jean_cook@med.unc.edu.
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004249:DNA Damage; D004331:Drosophila melanogaster; D064248:Geminin; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary
C103706:CDC6 protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; C577995:GMNN protein, human; D064248:Geminin; D009687:Nuclear Proteins