MUTATIONAL ANALYSIS OF THE CARBOXY-TERMINAL PORTION OF P53 USING BOTHYEAST AND MAMMALIAN-CELL ASSAYS IN-VIVO

Increasing evidence indicates that p53 is a transcriptional tuans-acti vator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the tuans-activation ability mainly due to loss of its sequence-specific DNA binding, Using both yeast and mammalian c ell assays, the effect of p53 mutations in the carboxy terminal portio n was investigated in order to address how p53 mutations outside of th e DNA binding domain affect p53 function, The p53 cDNA in the carboxy- terminus was randomly mutagenized by error-prone polymerase chain reac tions and the amplified cDNA was screened for the ability to trans-act ivate using a yeast assay, Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomeriza tion domain, were further analysed for trans-activation, cell cycle ar rest and colony formation in a human osteosarcoma cell line, Saos-2. T hese functional properties of p53 were disrupted by the missense mutat ions. Surprisingly, one of the nonsense mutations disrupts the trans-a ctivation function and the ability to G1 arrest but shows a strong inh ibition of colony formation, These results confirm that mutations in t he oligomerization domain can inactivate p53 function and also indicat e that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.