Polymerase Epsilon Is Required To Maintain Replicative Senescence

Abstract

Replicative senescence is a permanent cell cycle arrest in response to extensive telomere shortening. To understand the mechanisms behind a permanent arrest, we screened for factors affecting replicative senescence in budding yeast lacking telomere elongation pathways. Intriguingly, we found that DNA polymerase epsilon (Pol ) acts synergistically with Exo1 nuclease to maintain replicative senescence. In contrast, the Pol -associated checkpoint and replication protein Mrc1 facilitates escape from senescence. To understand this paradox, in which DNA-synthesizing factors cooperate with DNA-degrading factors to maintain arrest, whereas a checkpoint protein opposes arrest, we analyzed the dynamics of double-and single-stranded DNA (ssDNA) at chromosome ends during senescence. We found evidence for cycles of DNA resection, followed by resynthesis. We propose that resection of the shortest telomere, activating a Rad24Rad17 -dependent checkpoint pathway, alternates in time with an Mrc1-regulated Pol resynthesis of a short, double-stranded chromosome end, which in turn activates a Rad9 53BP1 -dependent checkpoint pathway. Therefore, instead of one type of DNA damage, different types (ssDNA and a double-strand break-like structure) alternate in a "vicious circle," each activating a different checkpoint sensor. Every time resection and resynthesis switches, a fresh signal initiates, thus preventing checkpoint adaptation and ensuring the permanent character of senescence.

Rif1 and Exo1 regulate the genomic instability following telomere losses

“…5D). The difference between checkpoint effects was most likely due to the essential role of Rad9 and Rad24 in maintaining replicative senescence (Deshpande et al ., 2011), whereas Chk1 may have a limited role. In conclusion, the critical role for Rif1 in cells lacking telomeres manifests when relevant checkpoint pathways are intact.…”

“…In contrast, Rad24 and Tel1 seem to function in the same pathway as Exo1. These data support the ‘Vicious Cycle’ model of replicative senescence, which stipulates that the continuously alternating activities of at least two pathways, involving Exo1‐Rad24 and Rad9‐Polymerase epsilon respectively, are required to maintain replicative senescence (Deshpande et al ., 2011). …”

“…To do so, they should be able to tolerate telomere losses and the associated genomic instability, and also to escape the DNA damage checkpoint control, through yet unknown mechanisms. One of the best models to identify such mechanisms is the PAL system, consisting of budding yeast cells unable to maintain telomeres, similarly to most human somatic cells (Maringele & Lydall, 2004b; Deshpande et al ., 2011). In this system, cells escape replicative senescence and proliferate indefinitely without telomeres, accumulating genomic deletions and amplifications, for example palindromic duplications (Maringele & Lydall, 2004b; Lee et al ., 2008), hence the name.…”

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…Such loss of fidelity can have major biological consequences, as seen by the development of cancer in mice which have lost the proofreading function of polymerase δ in vivo (Goldsby et al 2001). While there have yet been no studies directly linking nuclear DNA polymerase fidelity loss with ageing, 2 it is notable that polymerase ε is required to prevent replicative senescence in budding yeast that lack telomerase activity (Deshpande et al 2011). Moreover, instability of the nuclear genome can trigger apoptosis, neoplastic change or senescence, leading to organismal ageing.…”

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

“…Although the signal of senescence is not fully understood, it is believed that short telomeres lose their capping function and are subject to unregulated degradation by nucleases. Accordingly, the amount of single strand DNA increases at telomeres during senescence 31 and RPA is increasingly recruited to telomeres in parallel with the loss of viability of senescing cells 32 . Interestingly, the recruitment of RPA in regions adjacent to telomeres increases at the peak of senescence suggesting that degradation of the 5 0 strand reaches subtelomeric regions 32 .…”