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Abstract
The p53 tumor suppressor protein binds to DNA as a dimer of dimers to regulate transcription of genes that mediate responses to cellular stress. We have prepared a cross-linked trapped p53 core domain dimer bound to decamer DNA and have determined its structure by x-ray crystallography to 2.3A resolution. The p53 core domain subunits bind nearly symmetrically to opposite faces of the DNA in a head-to-head fashion with a loophelix motif making sequence-specific DNA contacts and bending the DNA by about 20 degrees at the site of protein dimerization. Protein subunit interactions occur over the central DNA minor groove and involve residues from a zinc-binding region. Analysis of tumor derived p53 mutations reveals that the dimerization interface represents a third hot spot for mutation that also includes residues associated with DNA contact and protein stability. Residues associated with p53 dimer formation on DNA are poorly conserved in the p63 and p73 paralogs, possibly contributing to their functional differences. We have used the dimeric protein-DNA complex to model a dimer of p53 dimers bound to icosamer DNA that is consistent with solution bending data and suggests that p53 core domain dimer-dimer contacts are less frequently mutated in human cancer than intra-dimer contacts.
Highlights
EXPERIMENTAL PROCEDURESMouse p53 Core Domain Purification—The mouse p53 core domain was purified essentially as described previously with minor modification [14]
With the consensus PuPuPuC(A/T)͉(A/T)GPyPyPy [7] with anywhere from 0 to 20 base pairs between them, an icosamer is more commonly found in vivo [8]
Implications for DNA binding by the p63 and p73 paralogs and for DNA binding by a p53 dimer of dimers are discussed
Summary
Mouse p53 Core Domain Purification—The mouse p53 core domain was purified essentially as described previously with minor modification [14]. Peak fractions containing p53DBD were pooled, concentrated, and further purified by gel filtration using a Superdex-75 preparative column (Amersham Biosciences). Preparation of the p53DBD Dimer-DNA Complex—Duplex DNA and protein were mixed in a 1:2 molar ratio in crosslinking buffer (20 mM sodium citrate, pH 6.1, 100 mM NaCl, and 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride) to a volume of 2 ml and incubated at 21 °C for 3 h. The cross-linked protein-DNA complex was purified by fast protein liquid chromatography gel filtration using a Superdex-75 preparative column. Crystallization, Data Collection, and Structure Determination—Crystals were prepared using hanging drop vapor diffusion against a reservoir containing 0.2 M NH4Cl, 0.01 M CaCl2, 0.05 M Tris-HCl, pH 8.5, 28% polyethylene glycol 4000 and appeared after 2 days, growing to a maximum size of 0.4 ϫ 0.05 ϫ 0.025 mm after 4 –5 days.
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