| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | SUMO4 knockout U2OSTrEx-FlpIn were generated using two different guide RNAs pSpCas9 (BB)-2A PURO (GenScript) plasmids that target the SUMO4 gene at nucleotides (relative to start codon) 150-171 (gRNA #1) and 186-204 (gRNA #4). 6xHis-FLAG SUMO2 K11R and K21R were generated by GenScript and cloned into pCDNA5/FRT/TO at BamHI and XhoI sites. Human SENP1 cDNA (ENST00000448372.5) was synthesized by GenScript to contain an N-terminal FLAG tag and synonymous siRNA resistance mutations to the exon 6 and 12 siRNA used (see key resources table, ‘Oligonucleotides’). 6xHis-HA-SUMO4 cDNA (GenBank: NM_001002255.1) was generated by GenScript to include synonymous mutations that render it insensitive to siRNA #2 and siRNA #3. 6xHis-HA-SUMO4 was subcloned into pcDNA5/FRT/TO using HindIII-BamHI sites. Human RAP80 (also known as UIMC1, ENST00000511320.6) was generated by gene synthesis (GenScript). The SIM binding mutant F40A/V41A/I42A was generated by SDM (GenScript). Mutations in Abraxas and BRCC36 were introduced by SDM (GenScript). | Get A Quote |
| Mutagenesis Services | Get A Quote | |
| PCR Cloning and Subcloning | Get A Quote | |
| Peptide Synthesis | Get A Quote |
The amplitudes of small-modifier protein signaling through ubiquitin and the small ubiquitin-like modifiers, SUMO1-3, are critical to the correct phasing of DNA repair protein accumulation, activity, and clearance and for the completion of mammalian DNA double-strand-break (DSB) repair. However, how SUMO-conjugate signaling in the response is delineated is poorly understood. At the same time, the role of the non-conjugated SUMO protein, SUMO4, has remained enigmatic. Here, we reveal that human SUMO4 is required to prevent excessive DNA-damage-induced SUMOylation and deleterious over-accumulation of RAP80. Mechanistically we show that SUMO4 acts independently of its conjugation and potentiates SENP1 catalytic ac... More
