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DCAF14 regulates CDT2 to promote SET8-dependent replication fork protection

Neysha Tirado-Class1, Caitlin Hathaway1, Anthony Nelligan1

  • 1Department of Molecular Biosciences, University of South Florida, Tampa, FL, USA.

Life Science Alliance
|November 8, 2023
PubMed

Insights

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.

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.

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