| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The PCR product was then used for site directed mutagenesis. MDC1, the longest isoform of human MDC1 (NM_014641.2) was used to generate synthetic MDC1 cDNA that was extensively codon optimised by GenScript to remove repetitive DNA sequences to enable gene synthesis...The K1840R mutation was made by GenScript...RNF4, human RNF4 (NM_002938.4) cDNA was synthesised by GenScript to contain resistance to two siRNA sequences, an N terminal HA tag, and cloned into pCDNA5/FRT/TO HindIII and BamHI sites...USP13 (NM_003940) was synthesised by GenScript to incorporate an N terminal HA tag, two sites of siRNA resistance and loss of BamHI and BglII sites by synonymous mutations...Streptavidin dynabeads (Invitrogen) were incubated with biotin conjugated SETDB1 peptides (GenScript, SETDB1SIMWT Biotin-RDSSSEDESSRPTEIIEIPDEDDD or SETDB1SIMMut BiotinRDSSSEDESSRPTEAAEAPDEDDD (100 ng per µl of bead slurry) for 1 hr in PBS supplemented with BSA. | Get A Quote |
SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as pa... More
