VI. Discussion
VI. 1 Non-canonical roles of p53 in tumour suppression
Tumour suppression by p53 was long thought to be a result of acute DNA damage responses including cell cycle arrest and repair. However, recent evidence suggests that p53 is indeed important for tumour suppression but its response to acute genotoxic stress is dispensable for this. Experimental evidence comes from knock-in mouse models carrying inactive TADs, revealing that losing the p5325,26 TAD1 impairs the ability to mediate cell cycle arrest and apoptosis but is still sufficient for tumour suppression, whereas a loss of both TADs in the p5325,26,53,54 mutant also impairs all three activities (Brady et al., 2011). A similar study by Li et al. demonstrated that p53 was still able to suppress tumour formation in mice when its DNA binding domain was mutated (Li et al., 2012). In addition, mice deficient for key effectors of the DNA damage response downstream of p53 (e.g. cdkn1a, puma, noxa) were not more prone to tumour formation than wildtype littermates (Valente et al., 2013). Taken together, this data highlights the importance of p53 in tumour suppression but suggests the likelihood of alternative pathways by which p53 achieves this.
Various non-canonical pathways for p53-mediated tumour suppression have been described in recent years (Mello and Attardi, 2018). Our work and others identified a role of p53 in maintaining genome integrity that further adds to its role as the “guardian of the genome” (Lane, 1992). This is achieved by transcriptional activation of target genes involved in DNA repair and by supporting DNA replication as well as restricting retrotransposon activity (Klusmann et al., 2016; Sengupta and Harris, 2005; Wylie et al., 2016; Yeo et al., 2016). Other studies have also attributed the title “guardian of the epigenome” to p53 as it seems to keep DNA methylation in check (Tovy et al., 2017).
Furthermore, p53 can also induce metabolic changes via non-canonical pathway. p53 inhibits both glycolysis and autophagy, processes involved in energy supply and the removal of damaged cellular components that are essential for tumour suppression (Kenzelmann Broz et al., 2013; Kruiswijk et al., 2015). In addition, p53 also inhibits stemness, promotes differentiation and is thought to regulate cell migration and invasion (Krizhanovsky and Lowe, 2009; Muller et al., 2011). Among others, these non-canonical roles of p53 contribute to its tumour suppressor activity but exactly how all these roles come together in a cell and whether they are activated in a cell type-/ or development-specific manner remains to be elucidated.
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VI.1.1 The non-canonical guardian of the genome supports DNA replication
Maintaining genome integrity is key to tumour suppression and seems to be mediated by p53 in a variety of ways. Next to the canonical pathways of the DNA damage response and checkpoints, we have identified a novel role of p53 in protecting a cell from endogenous DNA damage during replication. The presence of wildtype p53 in a cell supports DNA replication fork processivity that leads to a higher fork progression rate and a lower incidence of both fork stalling and extra origin firing (Fig.V.I.2). Manipulating the levels of p53 in a cell also modulates fork progression rates – an accumulation of p53 increases it, whereas a depletion of p53 reduces fork progression. As is the case for most other roles of p53, supporting DNA replication does not seem to be a direct effect of p53 but rather mediated via its transactivation of target genes. One argument for this is the delayed effect of p53 accumulation on fork progression. A three-hour treatment with the MDM2 inhibitor Nutlin-3a was not sufficient to affect replication even though p53 levels were elevated. In contrast, a longer incubation of cells with Nutlin-3a not only accumulated levels of p53 but also showed an increase in target gene expression, exemplified by p21 and MDM2, and in this time was able to increase replication fork progression significantly (Fig.V.I.2A-D, Suppl. Fig.V.I.2C-F). The fact that fork progression was not only modulated by p53 levels in U2OS cells that harbour high intrinsic replicative stress but also in primary non-transformed cells argues for the presence of a general role of p53 in supporting DNA replication that becomes even more important under replicative stress conditions found during tumourigenesis.
VI.1.2 A novel strategy to distinguish between fork velocity and fork processivity
In our study, we attempted to distinguish between replication fork velocity and processivity, both of which affect the length of the replicated tracks observed and thus the amount of under-replicated DNA and genome instability. Conventional fiber analysis solely assesses the length of two different labels in a given time and cannot distinguish between velocity and processivity of the DNA polymerase. The readout of a slow and a prematurely stalled replisome is the same – a short fiber and a low fork progression rate (Fig.VI.1.).
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Fig.VI.1 Fiber length is determined by both fork progression and processivity. cf. Suppl. Fig. V.I.7.3E Fiber length measured from labelled tracks in a conventional two-label Fiber assay is affected by both speed and processivity of the replication fork. (1) Fast polymerase movement allows more nucleotides to be incorporated, thus a longer labelled stretch appears. (2) Slow progression of the replication machinery results in shorter labelled tracks. (3) Fast but non-processive movement of replication also results in shorter labelled tracks as the label is not incorporated for the entire labelling time.
Conventional analysis of fork stalling involves the quantification of fibers with only the first label (red) incorporated. Mostly, the presence of red-only structures is accompanied by increased origin firing to compensate for fork stalling and can be analysed by quantifying green-red-green structures representing bidirectional first label origins. Quantification of these types of structures can provide an estimate of the amount of fork stalling and origin firing present in a sample but is not particularly accurate as these structures can also arise from different scenarios including replication termination that is not due to obstacles on the template but rather the end of a replicon.
To overcome limitations of the conventional assay, we developed a novel fiber assay strategy to distinguish between effects on fork velocity and processivity (Fig.V.I.2F-J;
Fig.V.II.2I-M). Analysis with both the conventional and the novel “fork stalling fiber assay”
revealed that p53 affects replication by supporting fork processivity rather than by increasing fork velocity. This effect was additionally accompanied by reduced origin firing seen in the conventional fiber assay. This complementing data suggests that the fork stalling fiber assay indeed works to dissect effects observed in the conventional fiber assay into fork velocity and processivity and can be used as an additional tool in future studies. However, a limitation of this method is the high number of samples that need to be analysed in order to obtain sufficient data and is therefore more suitable for providing additional information rather than for standard use in replication studies.
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An ongoing replication fork can be affected in many ways. On one hand the replisome itself can be altered by post-translational modifications and interaction partners to increase its association with the template DNA. Alternatively, the removal of obstacles along the DNA template such as proteins or secondary structures can also increase fork progression. Lastly, optimising the availability of building blocks in form of dNTPs would also be able to increase fork progression. As we only observe an effect of p53 on replication fork processivity and not velocity, the latter seems less likely to affect only one and not both parameters. Thus, a direct modification of the replisome or the loss of impediments on the DNA template seemed to be possible mechanisms by which p53 supports replication fork processivity.
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