Telomere length regulation by Rif1 protein from Hansenula polymorpha

Rif1 is a large multifaceted protein involved in various processes of DNA metabolism – from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension – a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.

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Cdc13 N-Terminal Dimerization, DNA Binding, and Telomere Length Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.00515-10 ◽

2010 ◽

Vol 30 (22) ◽

pp. 5325-5334 ◽

Cited By ~ 23

Author(s):

Meghan T. Mitchell ◽

Jasmine S. Smith ◽

Mark Mason ◽

Sandy Harper ◽

David W. Speicher ◽

...

Keyword(s):

Dna Binding ◽

Telomere Length ◽

Functional Characterization ◽

Point Mutations ◽

Telomeric Dna ◽

Dna Binding Domains ◽

Binding Domains ◽

Length Regulation ◽

Telomere Length Regulation

ABSTRACT The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

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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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Tel1 Activation by the MRX Complex Is Sufficient for Telomere Length Regulation but Not for the DNA Damage Response in Saccharomyces cerevisiae

Genetics ◽

10.1534/genetics.119.302713 ◽

2019 ◽

Vol 213 (4) ◽

pp. 1271-1288 ◽

Cited By ~ 4

Author(s):

Rebecca Keener ◽

Carla J. Connelly ◽

Carol W. Greider

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Telomere Length ◽

Dna Damage Response ◽

Telomere Elongation ◽

Link Type ◽

Epistasis Analysis ◽

Damage Response ◽

Length Regulation ◽

Telomere Length Regulation

Previous models suggested that regulation of telomere length in Saccharomyces cerevisiae by Tel1(ATM) and Mec1(ATR) would parallel the established pathways regulating the DNA damage response. Here, we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response, Rad53 is regulated by both Mec1 and Tel1. Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Our data suggest that nucleolytic telomere end processing is not a required step for telomerase to elongate telomeres.

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The Ku Heterodimer Performs Separable Activities at Double-Strand Breaks and Chromosome Termini

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8202-8215.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8202-8215 ◽

Cited By ~ 68

Author(s):

Alison A. Bertuch ◽

Victoria Lundblad

Keyword(s):

Telomere Length ◽

Large Subunit ◽

Double Strand Breaks ◽

Detailed Characterization ◽

Strand Breaks ◽

Functional Differences ◽

Length Regulation ◽

Telomere Length Regulation ◽

Dna Ends

ABSTRACT The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80tel alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80tel alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

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Tel1 activation by the MRX complex is sufficient for telomere length regulation but not for the DNA damage response in S. cerevisiae

10.1101/684522 ◽

2019 ◽

Author(s):

Rebecca Keener ◽

Carla J. Connelly ◽

Carol W. Greider

Keyword(s):

Dna Damage ◽

Telomere Length ◽

Dna Damage Response ◽

Mammalian Cells ◽

Telomere Elongation ◽

Epistasis Analysis ◽

Damage Response ◽

Length Regulation ◽

Processing Event ◽

Telomere Length Regulation

AbstractPrevious models suggested that regulation of telomere length in S. cerevisiae by Tel1(ATM) and Mec1(ATR) parallel the established pathways regulating the DNA damage response. Here we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response Rad53 is regulated by both Mec1 and Tel1. Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Since models that invoke a required end processing event for telomerase elongation are primarily based on the yeast pathways, our data call for a re-examination of the requirement for telomere end processing in both yeast and mammalian cells.

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Rif1 regulates telomere length through conserved HEAT repeats

Nucleic Acids Research ◽

10.1093/nar/gkab206 ◽

2021 ◽

Vol 49 (7) ◽

pp. 3967-3980

Author(s):

Calla B Shubin ◽

Rini Mayangsari ◽

Ariel D Swett ◽

Carol W Greider

Keyword(s):

Dna Binding ◽

Telomere Length ◽

Functional Role ◽

Conserved Residues ◽

Binding Interface ◽

Binding Function ◽

Length Regulation ◽

Telomere Length Regulation ◽

Telomere Regulation ◽

Conserved Amino Acids

AbstractIn budding yeast, Rif1 negatively regulates telomere length, but the mechanism of this regulation has remained elusive. Previous work identified several functional domains of Rif1, but none of these has been shown to mediate telomere length. To define Rif1 domains responsible for telomere regulation, we localized truncations of Rif1 to a single specific telomere and measured telomere length of that telomere compared to bulk telomeres. We found that a domain in the N-terminus containing HEAT repeats, Rif1177–996, was sufficient for length regulation when tethered to the telomere. Charged residues in this region were previously proposed to mediate DNA binding. We found that mutation of these residues disrupted telomere length regulation even when Rif1 was tethered to the telomere. Mutation of other conserved residues in this region, which were not predicted to interact with DNA, also disrupted telomere length maintenance, while mutation of conserved residues distal to this region did not. Our data suggest that conserved amino acids in the region from 436 to 577 play a functional role in telomere length regulation, which is separate from their proposed DNA binding function. We propose that the Rif1 HEAT repeats region represents a protein-protein binding interface that mediates telomere length regulation.

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Regulating telomere length from the inside out: The replication fork model

10.1101/041772 ◽

2016 ◽

Author(s):

Carol W Greider

Keyword(s):

Telomere Length ◽

Alternative Model ◽

Replication Fork ◽

Molecular Level ◽

Origin Firing ◽

Length Regulation ◽

Telomere Length Regulation ◽

Inside Out ◽

Counting Model

Telomere length is regulated around an equilibrium set point. Telomeres shorten during replication and are lengthened by telomerase. Disruption of the length equilibrium leads to disease, thus it is important to understand the mechanisms that regulate length at the molecular level. The prevailing protein counting model for regulating telomerase access to elongate the telomere does not explain accumulating evidence of a role of DNA replication in telomere length regulation. Here I present an alternative model: the replication fork model that can explain how passage of a replication fork and regulation of origin firing affect telomere length.

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