SUNY at Albany
June 19-23, 2001
Interaction of p53 to its natural promoters (waf1, and mdm-2) in the human genomic DNA; Implications of DNA structure and the oligomerization of p53 on its DNA binding specificity
p53 is a tumor suppressor protein. Its tumor suppressor activity is closely related to its DNA binding properties. It binds to a number of DNA response elements in the human genome and it is likely that these share common structural features. We have used atomic force microscopy in contact and magnetic/dynamic modes to investigate the structure of the nucleoprotein complexes containing p53 bound to several of its response elements in promoter regions of genes which it transactivates. Promoters examined include the waf1, RGC and mdm-2 found in the human genome. We show that these p53 binding sites although variable in their DNA sequence, have similar structural features including the bending of the helix axis when bound to p53. In addition, we show directly using microscopy that the oligomerization domain of p53 enhances the binding specificity of p53 to the target DNA (Fig 1).
Figure 1. Atomic force microscopy images of p53 (residues 94-360) bound to a 623 bp DNA fragmnet containing waf1 p53 response element.
A particularly interesting case involves promoters in which p53 binds to multiple sites located in proximity. We propose that DNA bending plays a special role in promoters of this type. Human mdm-2 gene has two p53 binding sites in its first intron separated by about two helical turns. The binding of p53 to both of these response elements has been shown to be essential for transactivation. This promoter region in the mdm-2 gene was PCR amplified from human genomic DNA. The complexes of this mdm-2 promoter DNA with wild type p53 and with several of its deletion mutants have been imaged using atomic force microscopy. DNA bending observed in the mdm-2 promoter region was found to be essential for the simultaneous binding of p53 to both mdm-2 response elements. As this human mdm-2 promoter region has been shown to be excluded from nucleosome formation, it is possible that the structural features of the mdm-2 promoter-p53 complexes reported here could have far reaching implications for in vivo p53 function in this gene.
P.Balagurumoorthy (1), Luda Shlyakhtenko (1), Kenneth M. Egan (1, 2), Stuart M. Lindsay (2) and Rodney E. Harrington (1)
Departments of Microbiology (1) and Physics and Astronomy (2),