OTUD5 limits replication fork instability by organizing chromatin remodelers.

Proper regulation of replication fork progression is important for genomic maintenance. Subverting the transcription-induced conflicts is crucial in preserving the integrity of replication forks. Various chromatin remodelers, such as histone chaperone and histone deacetylases are known to modulate replication stress, but how these factors are organized or collaborate are not well understood. Here we found a new role of the OTUD5 deubiquitinase in limiting replication stress. We found that OTUD5 is recruited to replication forks, and its depletion causes replication fork stress. Through its C-terminal disordered tail, OTUD5 assembles a complex containing FACT, HDAC1 and HDAC2 at replication forks. A cell line engineered to specifically uncouple FACT interaction with OTUD5 exhibits increases in FACT loading onto chromatin, R-loop formation, and replication fork stress. OTUD5 mediates these processes by recruiting and stabilizing HDAC1 and HDAC2, which decreases H4K16 acetylation and FACT recruitment. Finally, proteomic analysis revealed that the cells with deficient OTUD5-FACT interaction activates the Fanconi Anemia pathway for survival. Altogether, this study identified a new interaction network among OTUD5-FACT-HDAC1/2 that limits transcription-induced replication stress.

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

DDB1- and CUL4-associated factors (DCAFs) CDT2 and DCAF14 are substrate receptors for Cullin4-RING E3 ubiquitin ligase (CRL4) complexes. CDT2 is responsible for PCNA-coupled proteolysis of substrates CDT1, p21, and SET8 during S-phase of cell cycle. DCAF14 functions at stalled replication forks to promote genome stability, but the mechanism is unknown. We find that DCAF14 mediates replication fork protection by regulating CRL4CDT2 activity. Absence of DCAF14 causes increased proteasomal degradation of CDT2 substrates. When forks are challenged with replication stress, increased CDT2 function causes stalled fork collapse and impairs fork recovery in DCAF14-deficient conditions. We further show that stalled fork protection is dependent on CDT2 substrate SET8 and does not involve p21 and CDT1. Like DCAF14, SET8 blocks nuclease-mediated digestion of nascent DNA at remodeled replication forks. Thus, unregulated CDT2-mediated turnover of SET8 triggers nascent strand degradation when DCAF14 is absent. We propose that DCAF14 controls CDT2 activity at stalled replication forks to facilitate SET8 function in safeguarding genomic integrity.

PHIP variants associated with Chung-Jansen syndrome disrupt replication fork stability and genome integrity.

Chung-Jansen syndrome (CJS) is a rare, autosomal dominant disorder characterized by developmental delay, intellectual disability/cognitive impairment, behavioral challenges, obesity, and dysmorphic features. CJS is associated with heterozygous variants in PHIP (Pleckstrin-Homology Interacting Protein), a gene that encodes one of several substrate receptors for Cullin4-RING (CRL4) E3 ubiquitin ligase complex. Full length PHIP, also called DCAF14, was recently identified to function as a replication stress response protein. Herein, we report the identification of two PHIP missense variants identified by exome sequencing in unrelated individuals with CJS. The variants p.D488V and p.E963G occur in different functional elements of DCAF14- WD40 repeat domain and pleckstrin homology-binding region (PBR), respectively. Using DNA fiber assays, we reveal that cells expressing either variant exhibit defective replication fork progression in conditions of replication stress. Furthermore, unlike wild type DCAF14, both variants fail to accomplish DNA replication after exposure to genotoxic stress indicating a critical role of DCAF14 in protecting stalled replication forks. Thus, we have identified replication defects associated with CJS variants and predict replication-associated genome instability with CJS syndrome.

DCAF14 promotes stalled fork stability to maintain genome integrity.

Replication stress response ensures impediments to DNA replication do not compromise replication fork stability and genome integrity. In a process termed replication fork protection, newly synthesized DNA at stalled replication forks is stabilized and protected from nuclease-mediated degradation. We report the identification of DDB1- and CUL4-associated factor 14 (DCAF14), a substrate receptor for Cullin4-RING E3 ligase (CRL4) complex, integral in stabilizing stalled replication forks. DCAF14 localizes rapidly to stalled forks and promotes genome integrity by preventing fork collapse into double-strand breaks (DSBs). Importantly, CRL4DCAF14 mediates stalled fork protection in a RAD51-dependent manner to protect nascent DNA from MRE11 and DNA2 nucleases. Thus, our study shows replication stress response functions of DCAF14 in genome maintenance.