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. 2009 Apr 2;28(9):1246–1259. doi: 10.1038/emboj.2009.83

Figure 6.

Figure 6

Sumoylation of p53 in the DNA-bound state occurs at K386 and fails to dissociate p53 from its target sequence. (A) Sumoylation takes place on DNA-bound p53 without leading to its dissociation from the immobilized pWAFMLT template. Sumoylation reactions were performed with wild-type SUMO-1 or the GA mutant on p53- or 5KQ-bound pWAFMLT DNA as outlined. Protein-bound beads (B) and the unbound supernatant (UB) were analysed by Western blotting with anti-p53 full-length antibodies. (B) Sumoylation of DNA-bound p53 occurs at the same K386 residue. Sumoylation of wild-type p53 or the K292R or K386R mutant prebound to immobilized pWAFMLT DNA was performed and analysed as outlined. (C) Enhanced sumoylation on DNA-bound p53 fails to disengage p53 from DNA. DNA-bound p53 was subjected to different extent of sumoylation reactions with 2 times (2 × ) or 5 times (5 × ) more of sumoylation components. Binding assay was conducted as described in (A). (D) Recruitment of mSin3A but not p300 is enhanced by sumoylated p53 bound to DNA. HeLa nuclear extract was incubated with DNA-bound p53, with (+) or without (−) prior sumoylation reactions performed with wild-type (WT) or the GA mutant of SUMO-1. Proteins associated with DNA-bound p53 or sumoylated p53 were then analysed by Western blotting with anti-p53, p300 or mSin3A antibodies.