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PCR and Cloning> | Viruses were generated from 293T cells that were co-transfected with pMSCV-SDE2-Flag, pCMV-Gag/Pol and pCMV-VSV-G. Human UBR1 full-length cDNA was constructed by gene synthesis and cloned into pcDNA3-GFP KpnI-BamHI (Genscript). | Get A Quote |
Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2Ct, the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2Ct constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2Ct degradation upon UVC d... More