Intragenic Second Site Suppressor Mutations for p53 Cancer Mutations

Discovered in 1979 as a protein tightly bound to the large T antigen of the SV40 DNA tumor virus, p53 is now known for its cancer suppressor functions. p53 is a homo-tetrameric transcription factor that induces cell cycle arrest or apoptosis upon DNA damage, hypoxia, and other upstream signals. Approximately 50% of all human cancers contain mutations in the p53 gene, almost all of which are missense mutations in the core domain. Mutations can occur throughout the core domain, but there are six "hotspots" where cancer mutations occur most frequently. The long-term goal of the laboratory is to restore p53 function to mutant p53 protein because clinical studies have shown that patients with cancers containing wild-type p53 have a better prognosis than those containing mutated p53. Toward this challenging goal, the lab is currently identifying intragenic second site suppressor mutations to gain insights into how to stabilize the core domain of p53 mutants. The objective of my project was to identify such mutations that would override the deleterious effect of the hotspot cancer mutation Arg175His.

To find suppressor mutations, I used a functional p53 assay in yeast cells. The p53 cDNA is expressed under the control of the constitutive yeast promoter ADH1 on a plasmid containing the HIS3 marker gene and a CEN sequence. The HIS3 marker gene allows for the selection of the plasmid on SC-His plates, while the CEN sequence maintains the plasmid at one copy per cell. p53 binds to its consensus DNA binding site introduced upstream of URA3 and activates the reporter gene, thus resulting in a Ura⁺ phenotype.

To screen for suppressor mutations, I first "gapped" the expression plasmid for the p53 mutant Arg175His either upstream or downstream of the cancer mutation using restriction enzymes. Then I PCR-amplified under mutagenic conditions the regions overlapping with the gap. Both the gapped plasmids and the PCR products were then cotransformed into yeast cells that repaired the gapped plasmids by homologous recombination. The yeast cells were plated on SC-HisUra plates to select for intact plasmids and functional p53 protein. Once the Ura⁺ phenotype was determined to be plasmid-dependent, yeast colony PCR was performed to amplify the p53 region surrounding the Arg175His mutation. A diagnostic digestion with Tsp45 I enzyme then determined the presence of the cancer mutation. 7.3 x 10⁵ and 1.3 x 10⁷ transformants were screened for the upstream and downstream regions, yielding 68 and 161 His⁺Ura⁺ clones, respectively. Thus far, 206 clones have been analyzed further; only 2 clones for the downstream region have a plasmid-dependent Ura⁺ phenotype with a confirmed Arg175His mutation. These two clones are currently being pursued further. Additional screens for the upstream region, as well as screens mutagenizing the upstream and downstream regions simultaneously are planned.