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      Ku Binding on Telomeres Occurs at Sites Distal from the Physical Chromosome Ends

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          Abstract

          The Ku complex binds non-specifically to DNA breaks and ensures repair via NHEJ. However, Ku is also known to bind directly to telomeric DNA ends and its presence there is associated with telomere capping, but avoiding NHEJ. How the complex discriminates between a DNA break and a telomeric extremity remains unknown. Our results using a tagged Ku complex, or a chromosome end capturing method, in budding yeast show that yKu association with telomeres can occur at sites distant from the physical end, on sub-telomeric elements, as well as on interstitial telomeric repeats. Consistent with previous studies, our results also show that yKu associates with telomeres in two distinct and independent ways: either via protein-protein interactions between Yku80 and Sir4 or via direct DNA binding. Importantly, yKu associates with the new sites reported here via both modes. Therefore, in sir4Δ cells, telomere bound yKu molecules must have loaded from a DNA-end near the transition of non-telomeric to telomeric repeat sequences. Such ends may have been one sided DNA breaks that occur as a consequence of stalled replication forks on or near telomeric repeat DNA. Altogether, the results predict a new model for yKu function at telomeres that involves yKu binding at one-sided DNA breaks caused by replication stalling. On telomere proximal chromatin, this binding is not followed by initiation of non-homologous end-joining, but rather by break-induced replication or repeat elongation by telomerase. After repair, the yKu-distal portion of telomeres is bound by Rap1, which in turn reduces the potential for yKu to mediate NHEJ. These results thus propose a solution to a long-standing conundrum, namely how to accommodate the apparently conflicting functions of Ku on telomeres.

          Author Summary

          The Ku complex binds to and mediates the rejoining of two DNA ends that were generated by a double-stranded DNA break in the genome. However, Ku is known to be present at telomeres as well. If it would induce end-to-end joining there, it would create chromosome end-fusions that inevitably will lead to gross chromosome rearrangements and genome instability, common hallmarks for cancer initiation. Our results here show that Ku actually is associated with sites on telomeric regions that are distant from the physical ends of the chromosomes. We propose that this association serves to rescue DNA replication that has difficulty passing through telomeric chromatin. If so called one-sided breaks occur near or in telomeric repeats, they will generate critically short telomeres that need to be elongated. The binding of Ku may thus either facilitate the establishment of a specialized end-copying mechanism, called break induced replication or aid in recruiting telomerase to the short ends. These findings thus propose ways to potential solutions for the major conceptual problem that arose with the finding that Ku is associated with telomeres.

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          Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

          Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain. Copyright 1999 John Wiley & Sons, Ltd.
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            The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases.

            Homologs of the chromatin-bound yeast silent information regulator 2 (SIR2) protein are found in organisms from all biological kingdoms. SIR2 itself was originally discovered to influence mating-type control in haploid cells by locus-specific transcriptional silencing. Since then, SIR2 and its homologs have been suggested to play additional roles in suppression of recombination, chromosomal stability, metabolic regulation, meiosis, and aging. Considering the far-ranging nature of these functions, a major experimental goal has been to understand the molecular mechanism(s) by which this family of proteins acts. We report here that members of the SIR2 family catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assembly-linked and transcription-related acetyltransferases. Contrary to a recent report, we find no evidence that these proteins ADP-ribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes.
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              Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association.

              We determined the distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                8 December 2016
                December 2016
                : 12
                : 12
                : e1006479
                Affiliations
                [001]Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada
                Chinese Academy of Sciences, CHINA
                Author notes

                The authors have declared that no competing interests exist.

                • Conceptualization: MVL RSM RJW.

                • Formal analysis: MVL RSM RJW.

                • Funding acquisition: RJW.

                • Investigation: MVL EP RSM.

                • Methodology: MVL RSM.

                • Project administration: RJW.

                • Supervision: RJW.

                • Validation: MVL EP RSM.

                • Visualization: MVL EP.

                • Writing – original draft: MVL RJW.

                • Writing – review & editing: RJW.

                Author information
                http://orcid.org/0000-0002-1477-9006
                http://orcid.org/0000-0003-1131-4649
                http://orcid.org/0000-0001-6670-2759
                Article
                PGENETICS-D-16-01086
                10.1371/journal.pgen.1006479
                5145143
                27930670
                5bae77a9-19ee-4e06-80a3-447945c90dc5
                © 2016 Larcher et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 May 2016
                : 14 November 2016
                Page count
                Figures: 8, Tables: 0, Pages: 29
                Funding
                Funded by: CIHR Open
                Award ID: 97874
                Award Recipient :
                Funded by: CRCHUS
                Award Recipient :
                Funded by: Canada Reserch Chair for Telomere Biology
                Award Recipient :
                The work was supported by a grant from the Canadian Institutes of Health Research to RJW (CIHR #97874; http://www.cihr-irsc.gc.ca/e/193.html), the Canada Research Chair in Telomere Biology ( http://www.chairs-chaires.gc.ca/home-accueil-eng.aspx) to RJW, and the Centre de Recherche Clinique de l'Hôpital Universitaire de Sherbrooke (CRCHUS; http://cr.chus.qc.ca/) to RJW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromosomes
                Chromosome Structure and Function
                Telomeres
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromosomes
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Galactose
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Galactose
                Biology and life sciences
                Genetics
                DNA
                DNA replication
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA replication
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Biology and life sciences
                Genetics
                DNA
                DNA repair
                Non-Homologous End Joining
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA repair
                Non-Homologous End Joining
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Probe Techniques
                Probe Hybridization
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Probe Techniques
                Probe Hybridization
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Genetics
                Genetics

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