p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POL iota). These p53-POL iota complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53 beta, p53 gamma, Delta 40p53 alpha, Delta 133p53 alpha, and Delta 160p53 alpha differentially affect this p53-POL iota-dependent DDT pathway originally described for canonical p53 alpha. We show that the C-terminal isoforms p53 beta and p53 gamma, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POL iota recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Delta 133p53 alpha and Delta 160p53 alpha caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53 alpha exacerbated the DDT pathway defects, unveiling impaired POL iota recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53 alpha was particularly pronounced in cells co-expressing Delta 133p53 alpha or Delta 160p53 alpha. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53 alpha, carved out interference with p53 alpha functions in the p53-POL iota-dependent DDT pathway.