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Abstract 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. 

Abstract Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community. 

The gamma-glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout the multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M, S300F) whose combined heterozygosity causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation: complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX (fIX)) or calcification (Matrix Gla Protein (MGP)) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of VKD protein in the complex. The VKD protein in the complex with wild type carboxylase was carboxylated before challenge protein carboxylation occurred, and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time, and did not completely carboxylate fIX in the complex. S300F carboxylation was poor with both fIX and MGP. Additional studies analyzed fIX and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The V255M mutant generated more carboxylated peptide than wild type GGCX, however the peptides were partially carboxylated. Analysis of the V255M mutant in fIX-HEK293 cells lacking endogenous GGCX revealed poor fIX clotting activity. The studies show that disrupted processivity causes disease, and explain the defect in the patient. The kinetic analyses also suggest that disrupted processivity may occur in wild type carboxylase under some conditions, e.g. warfarin therapy or vitamin K deficiency. 

Telomeres cap chromosome ends with specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. Fission yeast telomeres can be formed by cleaving a “proto-telomere” bearing 48 bp of telomere repeats to form a new stable chromosomal end that prevents the rapid degradation seen at similar DNA double-strand breaks (DSBs). This end-protection was investigated in viable mutants lacking telomere-associated proteins. Telomerase, the shelterin components Taz1, Rap1, or Poz1 or the telomere-associated protein Rif1 were not required to form a stable chromosome end after cleavage of the proto-telomere. However, cells lacking the fission yeast shelterin component Ccq1 converted the cleaved telomere repeat-capped end to a rapidly degraded DSB. Degradation was greatly reduced by eliminating the nuclease activity of Mre11, a component of the Mre11-Rad50-Nbs1/Xrs2 complex that processes DSBs. These results demonstrate a novel function for Ccq1 to effect end-protection by restraining Mre11-dependent degradation.