Enhancement of p53 sequence-specific binding by DNA supercoiling

Abstract

Using a new competition assay, we investigated the effect of DNA negative supercoiling on the DNA sequencespecific binding (SSDB) of human wild-type (wt) p53 protein. We found that supercoiled (sc) pBluescript DNAs with different inserted p53 target sequences were stronger competitors than a mixture of scDNA pBluescript with the given 20-mer target oligodeoxynucleotide. ScDNAs were always better competitors than their linearized or relaxed forms. Two DNAs with extruded cruciforms within the target sequence were the best competitors; removal of the cruciforms resulted in a decrease of competitor strength. In contrast to the full-length wt p53, the deletion mutant p53CD30 and the p53 core domain (93-312 aa) showed no enhancement of p53 SSDB to scDNA, suggesting that, in addition to the p53 core domain, the C-terminal was involved in this binding. We conclude that cruciforms and DNA bends contribute to the enhancement of p53 SSDB to scDNA and that the DNA supercoiling is an important determinant in the p53 sequence-specific binding. Supercoiling may thus play a significant role in the complex p53-regulatory network.

Amino acids–nucleotides biomolecular recognition: from biological occurrence to affinity chromatography

“…Furthermore, because negatively supercoiled DNA is underwound, it exerts a force that helps separate the two strands of the helix, which is required during both replication and transcription (Clark, 2005). As previously described, a number of protein-DNA complexes only occur because the binding is promoted by negative supercoiling due to stabilization of writhing (Palecek et al, 2004). Recent studies suggest a possible relation between the p53 function and the DNA topology.…”

“…Recent studies suggest a possible relation between the p53 function and the DNA topology. Palecek et al (2004) hypothesized that the DNA supercoiling degree may play a significant role in the complex p53-regulatory network.…”

Transcriptional regulation by p53: one protein, many possibilities

“…In addition to small molecules, other factors, like the supercoiled nature of the DNA or local DNA bending, may facilitate p53-DNA interactions. 59,61 Importantly, it has been demonstrated earlier that there is a direct correlation between the stability of p53-DNA complexes and the ability of the DNA to be bent by the p53 DBD upon binding. 68 A summary of the above regulatory effectors is shown in Figure 1.…”

“…57,58 Though controversial, the importance of the CTD in p53 functioning has been continuing to reveal itself in a number of more recent publications. McKinney and Prives, 59 Fojta et al, 60 and Palecek et al 61 have shown that the CTD is important for binding to various non-linear DNAs. In another study, McKinney et al 62 have demonstrated the involvement of CTD in the ability of p53 to diffuse linearly on DNA.…”

HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53

“…In addition, NMR analysis has shown that wild-type and ⌬Cp53 are identical in conformation and that the CT does not interact with other p53 domains (Ayed et al 2001). In addition to this controversy, recent reports have supported the importance of the p53 CT for p53 binding to nonlinear DNA (McKinney and Prives 2002;Fojta et al 2004;McKinney et al 2004;Palecek et al 2004). Finally, CT modification is necessary for efficient promoter activation mediated by p53 (Liu et al 2004;McKinney et al 2004).…”