noncanonical dna motifs as transactivation targets by wild type and mutant p53不在经典里的dna图案由野生型和突变型p53 transactivation目标.pdfVIP

Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53 1,2 1 1,3 1 1 Jennifer J. Jordan , Daniel Menendez , Alberto Inga , Maher Nourredine , Douglas Bell , Michael A. Resnick1,2* 1 Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, United States of America, 2 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 Unit of Molecular Mutagenesis and DNA Repair, National Institute for Cancer Research, IST, Genoa, Italy Abstract Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surpr