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

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 CRL4^CDT2 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 CRL4^CDT2 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.