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DCAF14 regulates CDT2 to promote SET8-dependent replication fork protection.

Neysha Tirado-Class1, Caitlin Hathaway1, Anthony Nelligan1

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DCAF14 protein safeguards genome stability by controlling CDT2 activity at stalled replication forks. This prevents degradation of SET8, protecting nascent DNA and ensuring proper cell cycle progression.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genomics

Background:

  • DDB1- and CUL4-associated factors (DCAFs) like CDT2 and DCAF14 are crucial components of Cullin4-RING E3 ubiquitin ligase (CRL4) complexes.
  • CDT2 mediates PCNA-coupled proteolysis of cell cycle regulators, including CDT1, p21, and SET8.
  • DCAF14's role in replication fork protection and genome stability at stalled forks was previously uncharacterized.

Purpose of the Study:

  • To elucidate the mechanism by which DCAF14 promotes genome stability at stalled replication forks.
  • To investigate the regulation of CRL4CDT2 activity by DCAF14.
  • To identify the specific CDT2 substrates involved in DCAF14-mediated replication fork protection.

Main Methods:

  • CRISPR-Cas9 gene editing to generate DCAF14-deficient cells.
  • Western blotting to assess protein levels and degradation.
  • Immunofluorescence microscopy to visualize replication forks and DNA damage markers.
  • Analysis of nascent DNA synthesis and degradation.

Main Results:

  • DCAF14 deficiency leads to increased proteasomal degradation of CDT2 substrates, particularly SET8.
  • Absence of DCAF14 results in stalled replication fork collapse and impaired recovery under replication stress.
  • Stalled fork protection by DCAF14 is dependent on SET8, which prevents nuclease-mediated degradation of nascent DNA.

Conclusions:

  • DCAF14 regulates CRL4CDT2 activity at stalled replication forks, preventing excessive degradation of SET8.
  • SET8 functions to protect nascent DNA at remodeled replication forks, a process dependent on DCAF14.
  • DCAF14-mediated control of CDT2 activity is essential for SET8 function in safeguarding genomic integrity during replication stress.