Sequence Variation and p53 DNA Binding Figure 2. Detection of binding interactions between activated p53 and a set of multiplexed REs. A six-plex set of oligonucleotide- conjugated beads, carrying p53 binding sites (ConA, ConC, p21, PUMA, GADD45) and a negative control (WRNC) were incubated with nuclear extracts for 60 minutes, and p53 binding was measured. Nuclear extracts were from non-treated lymphoblast cells (NT) or cells treated with 1 mM doxorubicin (DOXO) for 24 hrs. Addition of a competing oligonucleotide demonstrates binding specificity. Relative binding was obtained for each RE tested. The split vertical scale allows display of data from high and low values. Values shown are the means for each bead type+/2SD (n = 3). doi:10.1371/journal.pgen.1000462.g002 data are corrected for bead-specific signal effects by measurement of a biotinylated reverse oligonucleotide on the same bead/oligo construct conjugated with phycoerythrin/streptavidin as described in the Materials and Methods. Binding values are relative to the biotin signal. The ConA sequence (black bar) showed appreciable binding (0.073+/20.0045) in the NT extract, and this level was ,30-fold greater than the negative control (WRNC: 0.0023+/ 20.0009). Relative to the NT values, nuclear extracts from DOXO-treated cells had a 17-fold increase for ConA (1.26+/ 20.089), an 8-fold increase for p21 (0.27+/20.023) and a 5-fold increase for weak-binding GADD45 (0.054+/20.006). The DOXO-induced ConA signal was ,180-fold greater than the DOXO negative control value. For GADD45 there was an 8-fold greater signal over the negative control, demonstrating the large dynamic range of the assay for both strong and weak binding sequences. To examine binding specificity, the bead set was presented to nuclear extracts (NT and DOXO) along with excess of oligonucleotide designed to directly compete for binding (Figure 2, Competing Oligo). Specificity for p53 binding was clearly demonstrated by abolishment of the signal in extracts from both NT and DOXO (see also Figures S2 and S3). An excess amount of a noncompeting oligonucleotide was used to maximize signal by blocking nonspecific binding (see optimization in Figures S2, S3). MAPD detected DOXO-activated p53 binding over a broad dynamic range (,2.5 logs) and nuclear extract concentra- tions (Figure S4). Using extracts with different quantities of p53 allows quantitative comparisons of biochemical properties (Table within Figure S4). To examine the general applicability of MAPD for measuring protein-DNA binding, we also created beads bearing different estrogen receptor a binding elements and measured ERa binding. ERa binding data is presented in the Supporting Information (Figure S5A,B). Impact of Sequence Variation on p53 Binding MAPD is highly suited to experiments such as site-directed mutagenesis of REs or SNP functional analysis, as shown in Figure 3A for p53 RE of p21, designated p21LWT. To investigate the importance of individual nucleotides on p53 binding, NT and DOXO-treated cell nuclear extracts were hybridized with 16 variants of the p21 RE in a 19-plex reaction that included two positive controls (ConA and p21LWT) and a negative control (WRNC). DOXO-treated cell extracts produced higher binding for all sequences. The p21 RE produced strong binding signals relative to WRNC, both when embedded in its native flanking sequence (p21LWT 0.62+/20.021) or an unrelated flanking sequence (p21GL3 0.58+/20.021; sequences are listed in Supporting Information). The perfect consensus p53 RE, ConA, showed a stronger signal (1.21+/20.083) relative to p21LWT. Changing the p21LWT sequence at nt-11 from C to G (Figure 3A 11C.G), a change favoring consensus, resulted in binding that was slightly higher (0.64+/20.038) than the native p21LWT. Moreover, when positions nt-11 and nt-20 in p21LWT were simultaneously changed to better match the consensus (Figure 3A, 11C.G; 20G.C), there was a 29% increase in p53 binding (from 0.64 to 0.83+/20.069). This provides an example of how PLoS Genetics | www.plosgenetics.org 3 May 2009 | Volume 5 | Issue 5 | e1000462