Abstract
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 (POLι). These p53-POLι 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 p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, 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 POLι 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, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. 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 p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.
Highlights
Already in the nineties, shortly after p53 was recognized to be a tumor suppressor [1, 2], it became clear that it is a key player in the protection of the genome integrity [3]
The p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α have lost p53α′s activity to stimulate replicationassociated recombination Our previous work demonstrated that p53α bypasses replication obstacles via a homology-directed DNA damage tolerance (DDT)-pathway which can be detected by measurements of spontaneous, i.e., replicationassociated recombination events [19]
While p53α-expressing cells stimulated p21 and to a lesser extent MDM2 expression in K562 cells, alternative p53-isoforms failed to induce p21 (Fig. 1B, C). Both N-terminally truncated and C-terminally modified p53-isoforms were impaired in activating homology-directed DDT
Summary
Shortly after p53 was recognized to be a tumor suppressor [1, 2], it became clear that it is a key player in the protection of the genome integrity [3]. TP53 gene products other than the canonical p53α lack N- and C-terminal domains of the human protein with welldefined biochemical functions like the first transcriptional transactivation domain (TAD1, amino acids [aa] 1 to 39 in p53α) in Δ40p53α (Fig. 1A). These alternative p53-isoforms were found to be differentially expressed in normal and cancer tissues, revealing pro-survival features of N-terminally truncated Δ133p53 and Δ160p53 in a p53α-dependent and -independent manner [15]. Co-expression of alternative p53-isoforms provides an additional mechanism to modulate p53αs tumor suppressor functions beyond TP53 gene mutations mostly affecting the DNAbinding domain (DBD) [17]
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