ABSTRACT
Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.
Subject(s)
DNA Replication , DNA, Single-Stranded , Humans , DNA, Single-Stranded/genetics , DNA Replication/genetics , Replication Protein A/genetics , Replication Protein A/metabolism , Protein Binding , Ubiquitination , DNA Damage , Genomic Instability , DNA Helicases/genetics , DNA Helicases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Recently, there have been increasing indications that the endocannabinoid (eCB) system is involved in vision. Multiple research teams studied the cannabinoid receptor type 2 (CB2R) expression and function in the mouse retina. Here, we examined the consequence of CB2R modulation on visual acuity using genetic and pharmacologic tools. We found that Cnr2 knockout mice show an enhanced visual acuity, CB2R activation decreased visual acuity while CB2R blockade with the inverse agonist AM630 increased it. The inhibition of 2-arachidonylglycerol (2-AG) synthesis and degradation also greatly increased and decreased visual acuity, respectively. No differences were seen when the cannabinoid receptor type 1 (CB1R) was deleted, blocked or activated implying that CB2R exclusively mediates cannabinoid modulation of the visual acuity. We also investigated the role of cannabinoids in retinal function using electroretinography (ERG). We found that modulating 2-AG levels affected many ERG components, such as the a-wave and oscillatory potentials (OPs), suggesting an impact on cones and amacrine cells. Taken together, these results reveal that CB2R modulates visual acuity and that eCBs such as 2-AG can modulate both visual acuity and retinal sensitivity. Finally, these findings establish that CB2R is present in visual areas and regulates vision-related functions.
