Plasmid Constructs for p53 and DNA Structures

Plasmids encoding human p53 proteins pT7-7wtp53 (full length wild type p53,p53, aa 1–393), pET-p53CD (p53CD, aa 94–312), pGEX-2TKp53CT (GST-p53CT, aa 320–393), pGEX-2TKp53T (GST-p53T, aa 363–393) and pGEX-4Tp53CD (GST-p53CD, aa 94–312) were described in [10 (link), 29 (link)]. Plasmids with T.A.T triplex forming sequences (pBA50 and pPA50) were prepared by cloning of (dT)₅₀.(dA)₅₀ into the EcoRV site of pBluescript SK II- (pBSK, Stratagene) and pPGM1 [34 (link)] (S1 Table). Similarly, plasmids for cruciform formation (pBAT34, pPAT34) were prepared by cloning (dAdT)₃₄ sequences to the same plasmids, for details see S1 Table. Plasmid pA69 with (dT)₆₉.(dA)₆₉ (on pUC19 basis [35 (link)]) and pUC19 control plasmid were used. Nonspecific competitor (pBSK/SmaI) was prepared by SmaI restriction enzyme (Takara, Japan) cleavage of pBSK. Plasmids for luciferase reporter assay (pGL3-BSK, pGL3-P1, pGL3-BA50, pGL3-PA50, pGL3-PA20_,S1 Table) were constructed by cloning fragments from pBSK derivatives into the SmaI/XhoI site of the pGL3-promoter (Invitrogen). All plasmids were isolated from bacterial strain TOP10 (Stratagene) and verified by sequencing.