Ku80 is involved in telomere maintenance but dispensable for genomic stability in Leishmania mexicana

Background Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. Methodology/Principal findings We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. Conclusion/Significance Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

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Cdc13 Cooperates with the Yeast Ku Proteins and Stn1 To Regulate Telomerase Recruitment

Molecular and Cellular Biology ◽

10.1128/mcb.20.22.8397-8408.2000 ◽

2000 ◽

Vol 20 (22) ◽

pp. 8397-8408 ◽

Cited By ~ 80

Author(s):

Nathalie Grandin ◽

Christelle Damon ◽

Michel Charbonneau

Keyword(s):

Positive Control ◽

Negative Control ◽

Telomere Maintenance ◽

Telomeric Dna ◽

Telomere Shortening ◽

Telomere Elongation ◽

Loading Function ◽

Ku Proteins ◽

Telomere Lengthening ◽

Direct Regulator

ABSTRACT The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both onSTN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.

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Genome stability is guarded by yeast Rtt105 through multiple mechanisms

Genetics ◽

10.1093/genetics/iyaa035 ◽

2021 ◽

Vol 217 (2) ◽

Author(s):

Yves Corda ◽

Laetitia Maestroni ◽

Pierre Luciano ◽

Maria Y Najem ◽

Vincent Géli

Keyword(s):

Genome Stability ◽

Large Subunit ◽

Telomere Maintenance ◽

Mobile Dna ◽

Yeast Saccharomyces Cerevisiae ◽

Genetic Analyses ◽

Telomere Elongation ◽

Defects Analysis ◽

In Cells

Abstract Ty1 mobile DNA element is the most abundant and mutagenic retrotransposon present in the genome of the budding yeast Saccharomyces cerevisiae. Protein regulator of Ty1 transposition 105 (Rtt105) associates with large subunit of RPA and facilitates its loading onto a single-stranded DNA at replication forks. Here, we dissect the role of RTT105 in the maintenance of genome stability under normal conditions and upon various replication stresses through multiple genetic analyses. RTT105 is essential for viability in cells experiencing replication problems and in cells lacking functional S-phase checkpoints and DNA repair pathways involving homologous recombination. Our genetic analyses also indicate that RTT105 is crucial when cohesion is affected and is required for the establishment of normal heterochromatic structures. Moreover, RTT105 plays a role in telomere maintenance as its function is important for the telomere elongation phenotype resulting from the Est1 tethering to telomeres. Genetic analyses indicate that rtt105Δ affects the growth of several rfa1 mutants but does not aggravate their telomere length defects. Analysis of the phenotypes of rtt105Δ cells expressing NLS-Rfa1 fusion protein reveals that RTT105 safeguards genome stability through its role in RPA nuclear import but also by directly affecting RPA function in genome stability maintenance during replication.

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RAD50 and RAD51 Define Two Pathways That Collaborate to Maintain Telomeres in the Absence of Telomerase

Genetics ◽

10.1093/genetics/152.1.143 ◽

1999 ◽

Vol 152 (1) ◽

pp. 143-152 ◽

Cited By ~ 12

Author(s):

Siyuan Le ◽

J Kent Moore ◽

James E Haber ◽

Carol W Greider

Keyword(s):

Cell Death ◽

Telomere Length ◽

Gene Conversion ◽

De Novo ◽

Dna Recombination ◽

Telomere Maintenance ◽

Triple Mutant ◽

Yeast Telomerase ◽

Telomere Lengthening

Abstract Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.

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Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00299.2012 ◽

2013 ◽

Vol 304 (12) ◽

pp. G1103-G1116 ◽

Cited By ~ 7

Author(s):

Shigeru B. H. Ko ◽

Sakiko Azuma ◽

Yukihiro Yokoyama ◽

Akiko Yamamoto ◽

Kazuhiro Kyokane ◽

...

Keyword(s):

Autoimmune Pancreatitis ◽

Genome Stability ◽

Progenitor Cell ◽

Es Cells ◽

Embryonic Stem ◽

Corticosteroid Treatment ◽

Human Pancreas ◽

Cell Markers ◽

Telomere Elongation

We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4+) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4+cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4+cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas.

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Role for the Related Poly(ADP-Ribose) Polymerases Tankyrase 1 and 2 at Human Telomeres

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.332-342.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 332-342 ◽

Cited By ~ 197

Author(s):

Brandoch D. Cook ◽

Jasmin N. Dynek ◽

William Chang ◽

Grigoriy Shostak ◽

Susan Smith

Keyword(s):

Potential Role ◽

Telomere Maintenance ◽

Telomeric Dna ◽

Continuous Growth ◽

Telomere Elongation ◽

Bona Fide ◽

Tankyrase 1 ◽

Growth Of Tumor

ABSTRACT Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.

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Ku and the Stability of the Genome

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724302201035 ◽

2002 ◽

Vol 2 (2) ◽

pp. 61-65 ◽

Cited By ~ 2

Author(s):

Anna A. Friedl

Keyword(s):

Double Strand Breaks ◽

Telomere Maintenance ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Cellular Processes ◽

Molecular Functions ◽

Ku Proteins ◽

The Stability ◽

Dependent Processes

Ku proteins are associated with a variety of cellular processes such as repair of DNA-double-strand breaks, telomere maintenance and retrotransposition. In recent years, we have learned a lot about their cellular and molecular functions and it has turned out that Ku-dependent processes affect the stability of the genome, both positively and negatively, in several ways. This article gives an overview on the role of Ku in determining the shape of the genome.

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Transcriptional activity of TRF2 is telomere length dependent

10.1101/056481 ◽

2016 ◽

Author(s):

Ananda Kishore Mukherjee ◽

Shalu Sharma ◽

Parashar Dhapola ◽

Dhurjhoti Saha ◽

Tabish Hussain ◽

...

Keyword(s):

Telomere Length ◽

Genome Stability ◽

Telomere Maintenance ◽

Regulatory Control ◽

Gene Promoters ◽

Multiple Gene ◽

Telomere Repeat ◽

Genome Wide ◽

Binding Factor

AbstractTRF2 is a telomere repeat binding factor crucial for telomere maintenance and genome stability. An emerging non-conventional role of TRF2 is as a transcriptional regulator through extra-telomeric bindings. Herein we report that increase in telomere length leads to sequestration of TRF2 at the telomeres leading to reduced extra-telomeric TRF2 occupancy genome wide. Decrease in TRF2 occupancy was found on multiple gene promoters in cells with elongated telomeres, including the cell cycle regulator kinase-p21. We found that TRF2 is a transcriptional repressor of p21, and, interestingly, TRF2-mediated regulatory control of p21 is telomere length dependent.

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The Role of Rif1 in telomere length regulation is separable from its role in origin firing

10.1101/2020.04.24.060491 ◽

2020 ◽

Author(s):

Calla B. Shubin ◽

Carol W. Greider

Keyword(s):

Telomere Length ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Origin Firing ◽

Replication Complex ◽

Telomere Elongation ◽

Length Regulation ◽

Telomere Length Regulation ◽

Telomere Lengthening

AbstractTo examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ΔN-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that deregulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-Δ1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

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Telomeres and Cancer

Life ◽

10.3390/life11121405 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1405

Author(s):

Hueng-Chuen Fan ◽

Fung-Wei Chang ◽

Jeng-Dau Tsai ◽

Kao-Min Lin ◽

Chuan-Mu Chen ◽

...

Keyword(s):

Telomere Length ◽

Molecular Mechanisms ◽

Telomere Maintenance ◽

Cancer Development ◽

Telomere Elongation ◽

Metabolism Pathway ◽

Length Regulation ◽

Telomere Length Regulation ◽

Genome Protection

Telomeres cap the ends of eukaryotic chromosomes and are indispensable chromatin structures for genome protection and replication. Telomere length maintenance has been attributed to several functional modulators, including telomerase, the shelterin complex, and the CST complex, synergizing with DNA replication, repair, and the RNA metabolism pathway components. As dysfunctional telomere maintenance and telomerase activation are associated with several human diseases, including cancer, the molecular mechanisms behind telomere length regulation and protection need particular emphasis. Cancer cells exhibit telomerase activation, enabling replicative immortality. Telomerase reverse transcriptase (TERT) activation is involved in cancer development through diverse activities other than mediating telomere elongation. This review describes the telomere functions, the role of functional modulators, the implications in cancer development, and the future therapeutic opportunities.

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The role of Rif1 in telomere length regulation is separable from its role in origin firing

eLife ◽

10.7554/elife.58066 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Calla B Shubin ◽

Carol W Greider

Keyword(s):

Telomere Length ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Origin Firing ◽

Replication Complex ◽

Telomere Elongation ◽

Length Regulation ◽

Telomere Length Regulation ◽

Telomere Lengthening

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that de-regulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

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