Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Biosciences

Major Professor

Huzefa Dungrawala, Ph.D.

Committee Member

Kristina Schimdt, Ph.D.

Committee Member

Sandy Westerheide, Ph.D.

Committee Member

Marcus Cooke, Ph.D.

Committee Member

Mark Alexandrow, Ph.D.

Keywords

Chung-Jansen Syndrome, Fork protection, Fork reversal, PHIP, Replication stress response

Abstract

The response to replication stress is critical for ensuring the stability of replication forks and preserving genome integrity. To aid this response, several factors are recruited to stalled replication forks to prevent irreversible collapse of the replication machinery. Replication fork proteomic analyses of stalled forks reveal enrichment of DCAF14, a component of the Cullin4-RING (CRL4) E3 ubiquitin ligase complex, that mediates replication fork protection by blocking nuclease-mediated digestion of nascent DNA. However, little is known about the molecular mechanism by which DCAF14 functions in the context of replication stress. Notably, loss of function mutations in DCAF14 are associated with a developmental disorder called Chung-Jansen syndrome but the causative functions that link DCAF14 to this syndrome remain poorly characterized. Herein, we demonstrate that the interaction of DCAF14 with the CRL4 complex is important to mediate stalled fork protection. Furthermore, characterization of PHIP variants from individuals with CJS syndrome demonstrates that the function of DCAF14 is perturbed in conditions of replication stress. We also show that DCAF14 exerts regulatory control over CRL4CDT2 activity, preventing the excessive degradation of CDT2 substrates. Consequently, the loss of DCAF14 culminates in the collapse of replication forks, particularly due to the dysregulation of the CDT2 substrate SET8, which protects nascent DNA at stalled replication forks. In summary, these studies elucidate the mechanism by which DCAF14 acts at stalled replication forks to uphold genomic integrity. These findings underscore the pivotal role of DCAF14 in upholding genomic integrity during the intricate process of DNA replication.

Download

Share

COinS