Sequence-dependent cooperative binding of p53 to DNA targets and its relationship to the structural properties of the DNA targets.
The prime mechanism by which p53 acts as a tumor suppressor is as a transcription factor regulating the expression of diverse downstream genes. The DNA-binding domain of p53 (p53DBD) interacts with defined DNA sites and is the main target for mutations in human primary tumors. Here, we show that the CWWG motif, found in the center of each consensus p53 half-site, is a key player in p53/DNA interactions. Gel-mobility-shift assays provide a unique opportunity to directly observe the various oligomeric complexes formed between p53DBD and its target sites. We demonstrate that p53DBD binds to p53 consensus sites containing CATG with relatively low cooperativity, as both dimers and tetramers, and with even lower cooperativity to such sites containing spacer sequences. p53DBD binds to sites containing CAAG and CTAG with measurable affinity only when imbedded in two contiguous p53 half-sites and only as tetramers (with very high cooperativity). There are three orders-of-magnitude difference in the cooperativity of interaction between sites differing in their non-contacted step, and further two orders-of-magnitude difference as a function of spacer sequences. By experimentally measuring the global structural properties of these sites, by cyclization kinetics of DNA minicircles, we correlate these differences with the torsional flexibility of the binding sites.
Department of Biology, Technion, Technion City, Haifa 32000, Israel.
Itai
I
Beno
Karin
K
Rosenthal
Michael
M
Levitine
Lihi
L
Shaulov
Tali E
TE
Haran
D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
D020033:Protein Isoforms; D016159:Tumor Suppressor Protein p53; D004247:DNA