| DNA replication is frequently challenged by both endogenous and exogenous genotoxic stress.These stresses,if not properly addressed,will lead to genomic instability and human diseases.Cells have evolved a number of mechanisms to counteract the genotoxic effects of DNA lesions.For instance,replication fork reversal is a common cellular response to replication stress and plays critical roles in preventing stalled replication forks from collapse.UFMylation is a newly identified ubiquitination-like protein modification.Similar to ubiquitination,UFMylation also utilizes a cascade three-enzyme system: UFM1-activating E1 enzyme UBA5,UFM1-conjugating E2 enzyme UFC1,and UFM1 E3 ligase UFL1.In this study,we show that UFL1-depleted cells are hypersensitive to replication stress-inducing agents,including HU and CPT.Moreover,depletion of UFL1 prevents the degradation of nascent DNA caused by BRCA2(Breast cancer type 2 susceptibility protein)inactivation in HU-treated cells.We further reveal that UFL1 interacts with PTIP(PAX-interacting protein 1)and this interaction is enhanced following HU treatment.Importantly,the first BRCT(BRCA1 C-terminus)domain of PTIP is essential for its interaction with UFL1 and their interaction is blocked by ATR(Ataxia telangiectasia and Rad3-related protein)inhibition.Given that PTIP is required for the recruitment of the MRE11(Meiotic recombination 11)nuclease to stalled replication forks,we speculate that ATR may phosphorylate UFL1 to promote its interaction with PTIP,thereby facilitating MRE11-dependent restart of stalled replication forks.In addition,by using tandem affinity purification approach to isolate UFL1-associaed protein complex,we identify several replication fork-associated proteins,including FANCI and FANCD2.We further demonstrate that FANCI and FANCD2 can be modified by UFM1.Our findings not only provide novel insights into the mechanisms that regulate replication fork stability,but also strengthen our knowledge on cancer research and therapy. |
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