Consequences of telomere replication failure: the other end-replication problem

Abstract

Telomeres are chromosome-capping structures that protect ends of the linear genome from DNA damage sensors. However, these structures present obstacles during DNA replication. Incomplete telomere replication accelerates telomere shortening and limits replicative lifespan. Therefore, continued proliferation under conditions of replication stress requires a means of telomere repair, particularly in the absence of telomerase. It was recently revealed that replication stress triggers break-induced replication (BIR) and mitotic DNA synthesis (MiDAS) at mammalian telomeres; however, these mechanisms are error prone and primarily utilized in tumorigenic contexts. In this review article, we discuss the consequences of replication stress at telomeres and how use of available repair pathways contributes to genomic instability. Current research suggests that fragile telomeres are ultimately tumor-suppressive and thus may be better left unrepaired.

Keywords: alternative lengthening of telomeres; break-induced replication; mitotic DNA synthesis; replication stress; telomeres.

Figure 1.. Replication Fork Obstacles within Telomeres.

Top, telomere replication primarily initiates from origins within the subtelomere and progresses to end of the chromosome. Bottom, the guanine-rich repetitive sequences and secondary structures within telomeres present obstacles to replisome progression during semi-conservative DNA replication, indicated by stars. Various proteins that transiently interact with shelterin as well as DNA and RNA maintenance pathways remove these obstacles to prevent replication fork stalling and collapse [94]. G quadruplexes are unwound by BLM and RTEL1 helicases [11, 44, 95]. Base lesions such as 8-oxoguanine are removed by base excision repair [3]. R-loops are prevented by nonsense mediated decay of TERRA and are dismantled by RNase H1 and the TERRA-interacting proteins NONO and SPFQ [96, 97]. t-loops are dismantled by RTEL1 [95].

Figure 2.. Consequences of Replication Fork Collapse within Telomeres.

A. Top, Replication fork collapse generates single-ended double-stranded breaks (DSBs) that cannot be repaired by direct ligation or conventional homology-directed repair. In telomeres, failure to restart replication at these break sites results in loss of unreplicated telomeric DNA, culminating in accelerated telomere shortening. Bottom, single-ended DSBs can complete replication through break-induced replication (BIR). Broken telomeres may utilize intra-, inter-, or extra-chromosomal telomeric sequences, potentially though annealing to microhomologies, to initiate long-tract BIR, resulting in conservative replication and unregulated telomere lengthening. B. Induced replication stress inhibits timely replication of fragile sites including CFSs and telomeres, causing cells to enter mitosis with unreplicated DNA and stalled replication fork intermediates. These sites are replicated by MiDAS detectable as mitotic EdU+ foci. MiDAS at CFSs and telomeres share common BIR-associated factors and are both dependent on the SLX4 nuclease scaffold protein, although CFS MiDAS requires the Mus81-EME1 nuclease while telomeric MiDAS does not. Telomeric MiDAS is almost exclusively replicated conservatively, while CFSs appear to partially rely on a semi-conservative replication mechanism.

Figure 3.. Cellular Response to Replication Stress at Telomeres.

Excessive replication stress causes replication fork stalling and collapse at origin-poor fragile sites such as telomeres. Left, noncancerous cells with normal cell cycle checkpoints do not attempt to repair telomeres that break during replication. This results in telomere loss and accelerated telomere shortening. If the nucleus accumulates five or more telomere dysfunction-induced foci (TIFs), cell cycle checkpoints will be activated to trigger senescence. Right, precancerous or cancerous cells with compromised cell cycle checkpoints restart telomere replication through break-induced replication (BIR) and mitotic DNA synthesis (MiDAS). Over the course of tumorigenesis, reliance on error-prone BIR generates additional replication stress that may contribute to cell transformation, alter telomeric chromatin compaction, and activate alternative lengthening of telomeres (ALT). In ALT+ cells, an altered BIR mechanism that acts in G2 and relies on telomere clustering in PML bodies (APBs) continuously repairs broken telomeres while introducing additional replication stress. This manifests a positive feedback loop that ultimately generates genome instability that threatens the viability of the cell and its host.