Summary Mammalian telomeres are associated with shelterin, the telomere specific protein complex that solves the end-protection problem. The telomeric shelterin binding sites, TTAGGG repeats, are maintained by telomerase, which solves the end-replication problem. We report that the TTAGGG repeat arrays of human and mouse telomeres challenge the DNA replication machinery, giving rise to replication-dependent defects that resemble those of the aphidicolin-induced common fragile sites. Conditional gene deletion experiments showed that efficient duplication of telomeric DNA requires the shelterin component TRF1. In the absence of TRF1, telomeres activate the ATR kinase in S phase and show a fragile site phenotype in metaphase. SMARD showed that TRF1 promotes efficient replication of TTAGGG repeats and prevents fork stalling. Two helicases that can remove G4 DNA structures, BLM and RTEL1, were required to repress the fragile telomere phenotype. These results identify a second telomere replication problem that is solved by the shelterin component TRF1.

The alternative nonhomologous end-joining (alt-NHEJ) machinery facilitates a number of genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions1,2,3. Using next-generation sequencing technology, we found that repair by alt-NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify Polymerase theta (Polθ; encoded by PolQ) as a critical alt-NHEJ factor in mammalian cells. PolQ inhibition suppresses alt-NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that PolQ loss results in increased rates of homology directed repair (HDR), evident by recombination of dysfunctional telomeres and accumulation of Rad51 at double stranded breaks. Lastly, we show that depletion of PolQ has a synergistic impact on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumors carrying mutations in HDR genes.

The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or non-homologous end-joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genomewide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1, limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage, and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.53BP1 can influence the type of DNA repair at DSBs (1) as seen in immunoglobulin gene rearrangements (2-4) and in the fusion of telomeres rendered dysfunctional through the removal of the shelterin protein TRF2 (5), where 53BP1 enhances the mobility of damaged telomeres, thus potentially promoting the chance of telomeretelomere encounters. In Brca1-deficient cells, 53BP1 enhances aberrant NHEJ events that create lethal radial chromosomes in response to poly(ADP-ribose) polymerase PARP1 inhibitors (PARPi) (6). In this setting, 53BP1 may favor NHEJ-mediated mis-rejoining by blocking the DSB resection needed for HDR (6, 7). 53BP1 was shown to impede 5' end resection at dysfunctional telomeres lacking all shelterin proteins and similarly, telomeres lacking only TRF2 show evidence of 53BP1-dependent protection from resection (5,8). Based on the finding that an allele of 53BP1 (53BP1 28A ) lacking all potential ATM/ATR kinase S/TQ target sites did not support immunoglobulin Class Switch Recombination (CSR) and failed to generate radial chromosomes in Brca1-deficient cells (7), it appears that these functions of 53BP1 involve interacting partner(s) modulated by the S/TQ sites. One candidate 53BP1-interacting factor is Rif1, which localizes to DSBs and dysfunctional telomeres, in a manner that is dependent on ATM signaling (9-11). Rif1 was originally identified as part of the telomeric complex in budding yeast (12) and was recently shown to inhibit resection at yeast telomeres (13,14). In contrast, mammalian Rif1 has no known function at functional telomeres but contributes to the intra-S phase checkpoint, facilitates recovery from replication stress, and affects replication timing (10,(15)(16)(17) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe introduced 53BP1 28A and other 53BP1 mutant alleles (7) into immortalized TRF2 F/-53BP1 -/-mouse embryo fibroblasts (MEFs) and induced telomere dysfunction by deletion of TRF2 (Fig. 1A,B). The results showed that the S/TQ sites were required for the accumulation of Rif1 at deprotected telomeres, whereas the GAR, BRCT, and oligomerization domains of 53BP1 were not ( Fig. 1A-C; fig. S1). The functional significance of the Rif1-53BP1 interaction was addressed using a tel...

The telomere end-protection problem is defined by the aggregate of DNA damage signaling and repair pathways that require repression at telomeres. To define the end-protection problem, we removed the whole shelterin complex from mouse telomeres through conditional deletion of TRF1 and TRF2 in nonhomologous end-joining (NHEJ) deficient cells. The data reveal two DNA damage response pathways not previously observed upon deletion of individual shelterin proteins. The shelterin-free telomeres are processed by microhomology-mediated alternative-NHEJ when Ku70/80 is absent and are attacked by nucleolytic degradation in the absence of 53BP1. The data establish that the end-protection problem is specified by six pathways [ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) signaling, classical-NHEJ, alt-NHEJ, homologous recombination, and resection] and show how shelterin acts with general DNA damage response factors to solve this problem.

DNA double-strand breaks (DSBs) disrupt the continuity of chromosomes and their repair by error-free mechanisms is essential to preserve genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism that involves alignment of microhomologous sequences internal to the broken ends before joining, and is associated with deletions and insertions that mark the original break site, as well as chromosome translocations. Whether MMEJ has a physiological role or is simply a back-up repair mechanism is a matter of debate. Here we review recent findings pertaining to the mechanism of MMEJ and discuss its role in normal and cancer cells.