Replication Stress as an Underappreciated Contribution to Cellular Senescence and Aging

Replication stress is the name given to disruptions to the process of DNA replication that takes place when a cell divides. The double stranded genome splits, unzipping into two single strands that are each provided with the complementary nucleotides in order to reform as two complete double-stranded copies. The rapidly moving point at which this unzipping takes place, where the two strands actually separate, is called the replication fork. It is a busy area of complex protein machinery, prone to failure and ongoing correction of failures. Unresolved failures lead to DNA damage, and DNA damage during this replication process can lead to cellular senescence.

Today's open access paper reviews what is known of the contribution of replication stress to the age-related burden of cellular senescence. Replication stress is a major culprit in at least one of the progeria conditions that give patients some of the appearance of accelerated aging, but to what degree is this the right place to look in order to measure the progression of normal aging? This is an open question, as replication stress is not so often measured versus markers of one of its outcomes, increased cellular senescence.

What other outcomes can replication stress produce in addition, however? Researchers here note an interesting connection to repair of double strand DNA breaks, that, as you may recall, has been implicated in driving the epigenetic changes that are characteristic of aging. If replication stress provokes greater double strand breaks and thus greater efforts to repair those breaks, it may well be a useful marker of aging.

Replication stress as a driver of cellular senescence and aging

Replication stress can be caused by an endogenous or environmental condition that disrupts the faithful copying of the genome. Replication stress is defined as stalling or slowing of replication fork progression which may lead to replication collapse and DNA damage. Stalled forks need to be protected and recovered to resume DNA synthesis and prevent genomic instability, a hallmark of aging.

A direct link between compromised stalled fork recovery and aging has been established by characterizing the molecular phenotypes of cells isolated from individuals with Werner Syndrome (WS), an autosomal recessive premature aging disease resulting from loss-of-function mutations in the WRN gene. Over the years, experimental evidence has demonstrated critical functions of the RECQ helicase WRN in stability and recovery of stalled replication forks under conditions of replication stress. Consistent with the roles of WRN in processing and stabilizing stalled forks, WS fibroblasts show reduced DNA replication capacity, fork asymmetry, heightened genomic instability, and premature replicative senescence. These phenotypes are likely attributed to failure to resolve complex replication intermediates resulting from stalled replication forks upon functional loss of WRN.

Cellular senescence driven by replication defects is considered a hallmark of aging. This prompts one to consider replicative stress as a potentially useful biomarker for aging. Although cell metabolism markers such as β-galactosidase staining have been a popular marker for senescent cells, markers of replication stress have not been as extensively studied. Rather, DNA damage emanating from replication stress or by other avenues (e.g., oxidative stress) has been postulated as a key biomarker for cellular senescence and even organismal aging. One of the most prominent DNA lesions associated with changes to the genome that is implicated in (and perhaps a driving force of) aging is the double strand break (DSB), one of the most lethal forms of DNA damage and a source of great genomic instability due to its recombinogenic nature.

Recently, the researchers developed an inducible DNA break mouse model that enabled them to investigate the importance of epigenetic changes induced by chromosome breaks for aging. Alterations in epigenetic landscape in regions surrounding the DSBs were associated with aging phenotypes at the cellular and organismal levels. However, whether the aging phenotypes associated with epigenetic changes are reversible at the organismal level remains to be seen. Nonetheless, the described model system will be useful for future work to study in vitro and in vivo aging. It remains to be determined if DSBs deriving from replication stress drive aging in replicative tissues by a mechanism that is different from the one described above, in which DSBs introduced frankly by the in vivo inducible restriction endonuclease system in both non-replicative and replicative tissues cause aging in a manner that is heavily dependent on epigenetic changes.

Although one could argue that DSBs represent only one of multiple DNA lesions to induce accelerated aging, the probability that they occur at the fork in replicative tissues in vivo is high. Replication fork stalling followed by blockage leads to single-stranded and ultimately DSBs, i.e., broken replication forks that cells must deal with using fork reconstruction pathways to preserve genomic stability. Typically, these repair mechanisms to heal DSBs at broken replication forks involve HR repair or the less faithful nonhomologous end-joining (NHEJ). Although stalled forks can be restarted by non-recombinogenic mechanisms, the transient single-stranded DNA that arises is susceptible to breakage. Thus, it is difficult to tease out if a structural feature of the stalled or arrested replication fork, the fork-associated DSB itself, or both represent a key signaling event in cellular senescence and aging. Either way, in proliferating cells of rapidly turning over tissues, replication stress is a driving force for age-associated signaling pathways associated with delayed fork progression.

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