Sequence-dependent cooperative binding of p53 to DNA targets and its relationship to the structural properties of the DNA targets
Sequence-dependent cooperative binding of p53 to DNA targets and its relationship to the structural properties of the DNA targets
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DOI：
10.1093/nar/gkq1044
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发表时间：
2011-03-01
影响因子：
14.9
通讯作者：
Haran, Tali E.
中科院分区：
文献类型：
--
作者：
Beno, Itai;Rosenthal, Karin;Haran, Tali E.
p53作为肿瘤抑制因子的主要机制是作为转录因子调节不同下游基因的表达。p53的DNA结合结构域（p53 DBD）与确定的DNA位点相互作用，并且是人类原发性肿瘤中突变的主要靶标。在这里，我们表明，CWWG基序，发现在每个共识p53半位点的中心，是p53/DNA相互作用的关键球员。凝胶迁移率变动分析提供了一个独特的机会，直接观察p53 DBD和其靶位点之间形成的各种寡聚复合物。我们证明，p53 DBD结合p53的共识网站含有CATG相对较低的协同性，作为二聚体和四聚体，甚至更低的协同性，这样的网站含有间隔序列。p53 DBD仅在嵌入两个连续的p53半位点中时以可测量的亲和力结合含有CAAG和CTAG的位点，并且仅作为四聚体（具有非常高的协同性）。在非接触步骤不同的位点之间的相互作用的协同性中存在三个数量级的差异，并且作为间隔序列的函数存在另外两个数量级的差异。通过实验测量这些网站的全局结构特性，通过DNA微环的环化动力学，我们将这些差异与结合位点的扭转灵活性相关联。
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.