STRAND ASYMMETRY OF CPG TRANSITIONS AS INDICATOR OF G(1) PHASE-DEPENDENT ORIGIN OF MULTIPLE TUMORIGENIC P53 MUTATIONS IN STEM-CELLS

In dividing cells, expression of mutations is DNA strand symmetric. Of all mutations originating de novo in nondividing cells, only those in the transcribed (noncoding) strand are immediately expressed in mRNA and protein. In contrast, any new mutation in the nontranscribed (codi ng) strand remains unexpressed until the cells enter S phase and begin proliferation. This previously unrecognized difference enables us to examine the cell cycle dependent origin of multiple tumorigenic mutati ons in stem cells. The human p53 gene, which acts as a gatekeeper in t he control of G(1) to S phase transition, was chosen for the analysis. Of all multiple mutations contained in p53 databases, we have tested in detail CpG transitions. Three features of CpG sites dictate this ch oice: C --> T transitions at methylated (m)CpG are the direct product of C-m deamination and are replication-independent; it is easy to iden tify the strand bearing a primary C-m --> T event because C --> T on t he transcribed strand appears as G --> A on the nontranscribed strand; and CpG transitions are the most frequent las both singular and multi ple occurrences) tumor-related p53 mutations. The origin of double non silent CpG transitions in nondividing cells predicts a significant exc ess of the heterostrand (C --> T, G --> A) doublets over the homostran d (C --> T, C --> T and G --> A, G --> A) doublets. For p53, we found such an excess. Based on this result, along with the results of three other tests reported here, we conclude that the majority of multiple p 53 mutations from human tumors occurred in quiescent stem cells.