Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

A proto-telomere is elongated by telomerase in a shelterin-dependent manner in quiescent fission yeast cells.

Telomere elongation is coupled with genome replication, raising the question of the repair of short telomeres in post-mitotic cells. We investigated the fate of a telomere-repeat capped end that mimics a single short telomere in quiescent fission yeast cells. We show that telomerase is able to elongate this single short telomere during quiescence despite the binding of Ku to the proto-telomere. While Taz1 and Rap1 repress telomerase in vegetative cells, both shelterin proteins are required for efficient telomere extension in quiescent cells, underscoring a distinct mode of telomerase control. We further show that Rad3ATR and Tel1ATM are redundantly required for telomere elongation in quiescence through the phosphorylation of Ccq1 and that Rif1 and its associated-PP1 phosphatases negatively regulate telomerase activity by opposing Ccq1 phosphorylation. The distinct mode of telomerase regulation in quiescent fission yeast cells may be relevant to that in human stem and progenitor cells.

RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand.

In Saccharomyces cerevisiae, the absence of Pif1 helicase induces the instability of G4-containing CEB1 minisatellite during leading strand but not lagging strand replication. We report that RPA and Pif1 cooperate to maintain CEB1 stability when the G4 forming strand is either on the leading or lagging strand templates. At the leading strand, RPA acts in the same pathway as Pif1 to maintain CEB1 stability. Consistent with this result, RPA co-precipitates with Pif1. This association between Pif1 and RPA is affected by the rfa1-D228Y mutation that lowers the affinity of RPA in particular for G-rich single-stranded DNA. At the lagging strand, in contrast to pif1Δ, the rfa1-D228Y mutation strongly increases the frequency of CEB1 rearrangements. We explain that Pif1 is dispensable at the lagging strand DNA by the ability of RPA by itself to prevent formation of stable G-rich secondary structures during lagging strand synthesis. Remarkably, overexpression of Pif1 rescues the instability of CEB1 at the lagging strand in the rfa1-D228Y mutant indicating that Pif1 can also act at the lagging strand. We show that the effects of the rfa1-D228Y (rpa1-D223Y in fission yeast) are conserved in Schizosaccharomyces pombe. Finally, we report that RNase H1 interacts in a DNA-dependent manner with RPA in budding yeast, however overexpression of RNase H1 does not rescue CEB1 instability observed in pif1Δ and rfa1-D228Y mutants. Collectively these results add new insights about the general role of RPA in preventing formation of DNA secondary structures and in coordinating the action of factors aimed at resolving them.

The inhibition of checkpoint activation by telomeres does not involve exclusion of dimethylation of histone H4 lysine 20 (H4K20me2).

DNA double-strand breaks (DSBs) activate the DNA damage checkpoint machinery to pause or halt the cell cycle.  Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints.  This "checkpoint privileged" status of telomeres was previously investigated in the yeast  Schizosaccharomyces pombelacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1.  However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin.  In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1.  Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined.

A Heterochromatin Domain Forms Gradually at a New Telomere and Is Dynamic at Stable Telomeres.

Heterochromatin domains play important roles in chromosome biology, organismal development, and aging, including centromere function, mammalian female X chromosome inactivation, and senescence-associated heterochromatin foci. In the fission yeast Schizosaccharomyces pombe and metazoans, heterochromatin contains histone H3 that is dimethylated at lysine 9. While factors required for heterochromatin have been identified, the dynamics of heterochromatin formation are poorly understood. Telomeres convert adjacent chromatin into heterochromatin. To form a new heterochromatic region in S. pombe, an inducible DNA double-strand break (DSB) was engineered next to 48 bp of telomere repeats in euchromatin, which caused formation of a new telomere and the establishment and gradual spreading of a new heterochromatin domain. However, spreading was dynamic even after the telomere had reached its stable length, with reporter genes within the heterochromatin domain showing variegated expression. The system also revealed the presence of repeats located near the boundaries of euchromatin and heterochromatin that are oriented to allow the efficient healing of a euchromatic DSB to cap the chromosome end with a new telomere. Telomere formation in S. pombe therefore reveals novel aspects of heterochromatin dynamics and fail-safe mechanisms to repair subtelomeric breaks, with implications for similar processes in metazoan genomes.

Eroded telomeres are rearranged in quiescent fission yeast cells through duplications of subtelomeric sequences.

While the mechanisms of telomere maintenance has been investigated in dividing cells, little is known about the stability of telomeres in quiescent cells and how dysfunctional telomeres are processed in non-proliferating cells. Here we examine the stability of telomeres in quiescent cells using fission yeast. While wild type telomeres are stable in quiescence, we observe that eroded telomeres were highly rearranged during quiescence in telomerase minus cells. These rearrangements depend on homologous recombination (HR) and correspond to duplications of subtelomeric regions. HR is initiated at newly identified subtelomeric homologous repeated sequences (HRS). We further show that TERRA (Telomeric Repeat-containing RNA) is increased in post-mitotic cells with short telomeres and correlates with telomere rearrangements. Finally, we demonstrate that rearranged telomeres prevent cells to exit properly from quiescence. Taken together, we describe in fission yeast a mode of telomere repair mechanism specific to post-mitotic cells that is likely promoted by transcription.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.