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Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non-homologous end joining (NHEJ) is upregulated, taz1? cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23MRE11-Rad50-Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1? lethality in G1. Neither Tel1ATM activation nor 5?-end resection was required for telomere fusion. Nuclease activity of Rad32MRE11 was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32MRE11 mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1? in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split-molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ. [ABSTRACT FROM AUTHOR]

Telomere protection and maintenance are accomplished through the coordinated actions of telomere-specific DNA binding proteins and their interacting partners. The fission yeast ortholog of human TRF1/2, Taz1, binds telomeric DNA and regulates numerous aspects of telomere function. Here, we ask which aspects of Taz1 function are mediated through its interacting proteins, Rap1 and Rif1. We demonstrate that rap1+ deletion phenocopies some, but not all, aspects of taz1Δ telomere dysfunction, while Rif1 exhibits a very different functional spectrum. Rap1 acts in a Taz1-dependent pathway to prevent chromosome end fusions and regulate telomeric 3′ overhang formation, while Rif1 is dispensable for these functions. Telomerase inhibition by Taz1 is mediated by two separate pathways, one involving Rap1 and the other involving Rif1. In contrast, Taz1 is uniquely required to prevent chromosomal entanglements and missegregation at cold temperatures. Strikingly, while rap1+ deletion exacerbates the cold sensitivity of taz1Δ cells, rif1+ deletion restores full viability. Thus, Rap1 and Rif1 are each required for a subset of the functions of Taz1, but each acquires Taz1-independent functions in its absence. Furthermore, Taz1 can function independently of its known binding partners. [ABSTRACT FROM AUTHOR]

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)‐Stn1‐Ten1, a telomeric ssDNA‐binding complex homologous to RPA. Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB‐fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3′‐ssDNA overhangs, indicative of defective lagging‐strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging‐strand DNA synthesis, thus terminating the cycle of telomere replication. Synopsis: The conserved (CTC1)‐Stn1‐Ten1 complex regulates DNA lagging strand synthesis and telomerase activity. CDK1‐dependent phosphorylation prevents Stn1 association with telomeres and is reversed by Ssu72 phosphatase, thereby allowing telomere overhang fill‐in reaction and consequently telomerase inhibition. A genome‐wide screen identifies Ssu72 as negative regulator of telomere elongation in fission yeastSsu72 is required for semiconservative DNA replication by controlling Stn1‐Pol α interactionSsu72 regulates Stn1 phosphorylation in fission yeast at a novel and highly conserved phosphorylation site located at the OB fold domain.The role of Ssu72 phosphatase in controlling telomere elongation via Stn1 recruitment is conserved in human cells. Reversal of inhibitory CDK phosphorylation on its Stn1 subunit allows (C)ST complex recruitment for telomeric DNA fill‐in synthesis in both fission yeast and human cells. [ABSTRACT FROM AUTHOR]