MaPS identifies <i>RAP1</i> as the gene whose product interacts with the Rap1 binding site.

<p>The yeast genomic DNA phage display library was selected for three rounds against a double-stranded oligonucleotide and PCR products of an upstream region containing Rap1 binding sites. The selected population of phage were profiled through microarray hybridization. Displayed is the distribution of the mean percentile rank for five independent such selections performed. The ORF corresponding to Rap1 had the highest mean percentile rank out of a total of 6242 ORFs queried on the array.</p

The Dot6 protein is a sequence-specific PAC element binding factor.

<p>Gel shift assay was performed with Dot6 DNA-binding domain and DNA containing PAC elements. Purified recombinant GST-Dot6 was incubated with 50 fM biotinylated probe containing 4 copies of the PAC element (bPAC4). Unbiotinylated competitor (PAC4) had identical sequence to the probe, or mutations in each copy of the PAC element (XPAC4).</p

The Rap1 DNA-binding domain is enriched in a sequence-specific and salt-dependent manner.

<p>A phage display library was affinity selected against indicated quantities of double-stranded DNA containing Rap1 binding sites under indicated salt conditions. Results from PCR of input phage library (input) or after second round of selection are shown. The intensity of a band is proportional to its abundance in the library. Lanes designated RAP1 indicate results of specific PCR against a single phage with the <i>RAP1</i> DNA-binding domain known to be in the library and the red arrows mark the expected size of this clone. Gel-isolation and sequencing of the selected bands at this location confirmed that they correspond to this clone. The blue arrows point out the PCR products corresponding to the <i>MCM1</i> DNA-binding domain (lower band) and <i>MCM1</i> ORF (upper band). The remaining bands show variable enrichment as a function of salt concentration and likely represent non-specific enrichment during the selection.</p

An overview of Microarray profiling of phage-display selection technology.

<p>(A) 1–3 kb fragments of yeast genomic DNA are cloned into T7 bacteriophage to create a translational fusion between the capsid protein and the peptide sequence encoded by the insert. (B) The library of phage are exposed to immobilized target DNA molecules and non-binding phage are washed away. Bound phage are eluted, amplified in liquid culture, and the process is repeated over multiple rounds. The sequence content of the enriched phage population is determined by PCR amplification of the inserts, labeling, and hybridization to a yeast ORF microarray.</p

Dot6 binds to PAC-containing promoters in vivo.

<p>Chromatin immunoprecipitation was performed on extracts of strain Y3648 (TAP-Dot6) and B4741 (untagged) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000449#s4" target="_blank">Materials and Methods</a>. Quantification of these data provided the percent of input DNA for the promoters of each of the indicated genes recovered in the immunoprecipitate. Shown are the fold enrichment of those values relative to the percent input DNA for the <i>ACT1</i> promoter recovered in the same immunoprecipitate. PCR quantification was performed in triplicate with less than 20% variation among replicates of individual samples.</p