Telomere Resolution Research Articles

Structure analysis of the telomere resolvase from the Lyme disease spirochete Borrelia garinii reveals functional divergence of its C-terminal domain.

Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two of the most common tick-borne illnesses. A characteristic feature of these spirochetes is their highly segmented genomes which consists of a linear chromosome and a mixture of up to approximately 24 linear and circular extrachromosomal plasmids. The complexity of this genomic arrangement requires multiple strategies for efficient replication and partitioning during cell division, including the generation of hairpin ends found on linear replicons mediated by the essential enzyme ResT, a telomere resolvase. Using an integrative structural biology approach employing advanced modelling, circular dichroism, X-ray crystallography and small-angle X-ray scattering, we have generated high resolution structural data on ResT from B. garinii. Our data provides the first high-resolution structures of ResT from Borrelia spirochetes and revealed active site positioning in the catalytic domain. We also demonstrate that the C-terminal domain of ResT is required for both transesterification steps of telomere resolution, and is a requirement for DNA binding, distinguishing ResT from other telomere resolvases from phage and bacteria. These results advance our understanding of the molecular function of this essential enzyme involved in genome maintenance in Borrelia pathogens.

Design and characterization of hyperactive mutants of the Agrobacterium tumefaciens telomere resolvase, TelA.

Telomere resolvases are a family of DNA cleavage and rejoining enzymes that produce linear DNAs terminated by hairpin telomeres from replicated intermediates in bacteria that possess linear replicons. The telomere resolvase of Agrobacterium tumefaciens, TelA, has been examined at the structural and biochemical level. The N-terminal domain of TelA, while not required for telomere resolution, has been demonstrated to play an autoinhibitory role in telomere resolution, conferring divalent metal responsiveness on the reaction. The N-terminal domain also inhibits the competing reactions of hp telomere fusion and recombination between replicated telomere junctions. Due to the absence of the N-terminal domain from TelA/DNA co-crystal structures we produced an AlphaFold model of a TelA monomer. The AlphaFold model suggested the presence of two inhibitory interfaces; one between the N-terminal domain and the catalytic domain and a second interface between the C-terminal helix and the N-core domain of the protein. We produced mutant TelA's designed to weaken these putative interfaces to test the validity of the modeled interfaces. While our analysis did not bear out the details of the predicted interfaces the model was, nonetheless, extremely useful in guiding design of mutations that, when combined, demonstrated an additive activation of TelA exceeding 250-fold. For some of these hyperactive mutants stimulation of telomere resolution has also been accompanied by activation of competing reactions. However, we have also characterized hyperactive TelA mutants that retain enough autoinhibition to suppress the competing reactions.

The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formation.

The telomere resolvase, TelA, forms the hairpin telomeres of the linear chromosome of Agrobacterium tumefaciens in a process referred to as telomere resolution. Telomere resolution is a unique DNA cleavage and rejoining reaction that resolves replicated telomere junctions into a pair of hairpin telomeres. Telomere resolvases utilize a reaction mechanism with similarities to that of topoisomerase-IB enzymes and tyrosine recombinases. The reaction proceeds without the need for high-energy cofactors due to the use of a covalent, enzyme-cleaved DNA intermediate that stores the bond energy of the cleaved bonds in 3'-phosphotyrosyl linkages. The cleaved DNA strands are then refolded into a hairpin conformation and the 5'-OH ends of the refolded strands attack the 3'-phosphotyrosine linkages in order to rejoin the DNA strands into hairpin telomeres. Because this kind of reaction mechanism is, in principle, reversible it is unclear how TelA controls the direction of the reaction and propels the reaction to completion. We present evidence that TelA forms and/or stabilizes a pre-cleavage intermediate that features breakage of the four central basepairs between the scissile phosphates prior to DNA cleavage to help propel the reaction forwards, thus preventing abortive cleavage and rejoining cycles that regenerate the substrate DNA. We identify eight TelA sidechains, located in the hairpin-binding module and catalytic domains of TelA, implicated in this process. These mutants were deficient for telomere resolution on parental replicated telomere junctions but were rescued by introduction of substrate modifications that mimic unwinding of the DNA between the scissile phosphates.

The Novel Yersinia enterocolitica Telomere Phage vB_YenS_P840 Is Closely Related to PY54, but Reveals Some Striking Differences.

Telomere phages are a small group of temperate phages, whose prophages replicate as a linear plasmid with covalently closed ends. They have been isolated from some Enterobacteriaceae and from bacterial species living in aquatic environments. Phage PY54 was the first Yersinia (Y.) enterocolitica telomere phage isolated from a nonpathogenic O:5 strain, but recently a second telomeric Yersinia phage (vB_YenS_P840) was isolated from a tonsil of a wild boar in Germany. Both PY54 and vB_YenS_P840 (P840) have a siphoviridal morphology and a similar genome organization including the primary immunity region immB and telomere resolution site telRL. However, whereas PY54 only possesses one prophage repressor for the lysogenic cycle, vB_YenS_P840 encodes two. The telRL region of this phage was shown to be processed by the PY54 protelomerase under in vivo conditions, but unlike with PY54, a flanking inverted repeat was not required for processing. A further substantial difference between the phages is their host specificity. While PY54 infects Y. enterocolitica strains belonging to the serotypes O:5 and O:5,27, vB_YenS_P840 exclusively lyses O:3 strains. As the tail fiber and tail fiber assembly proteins of the phages differ significantly, we introduced the corresponding genes of vB_YenS_P840 by transposon mutagenesis into the PY54 genome and isolated several mutants that were able to infect both serotypes, O:5,27 and O:3.

The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities

Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein–protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.

The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation.

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn’t recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3–6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3–6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3–6 hairpin than the wild-type hairpin. The SΔ1Δ3–6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30–37 days, while mice infected with SΔ1Δ3–6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV “flip and flop” telomeres are arranged.

Establishment of a near-contiguous genome sequence of the citric acid producing yeast Yarrowia lipolytica DSM 3286 with resolution of rDNA clusters and telomeres.

Yarrowia lipolytica is an oleaginous yeast that is particularly suitable for the sustainable production of secondary metabolites. The genome of this yeast is characterized by its relatively large size and its high number of different rDNA clusters located in its telomeric regions. However, due to the presence of long repetitive elements in the sub-telomeric regions, rDNA clusters and telomeres are missing in current genome assemblies of Y. lipolytica. Here, we present the near-contiguous genome sequence of the biotechnologically relevant strain DSM 3286. We employed a hybrid assembly strategy combining Illumina and nanopore sequencing reads to integrate all six rDNA clusters as well as telomeric repeats into the genome sequence. By fine-tuning of DNA isolation and library preparation protocols, we were able to create ultra-long reads that not only contained multiples of mitochondrial genomes but also shed light on the inter- and intra-chromosomal diversity of rDNA cluster types. We show that there are ten different rDNA units present in this strain that additionally appear in a predefined order in a cluster. Based on single reads, we also demonstrate that the number of rDNA repeats in a specific cluster varies from cell to cell within a population.

Single stranded DNA annealing is a conserved activity of telomere resolvases.

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA’s annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

The structure of human CST reveals a decameric assembly bound to telomeric DNA.

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.

Genomic characterization of two Orf virus isolates from Jilin province in China.

Orf virus (ORFV), a typical member of the Parapoxvirus genus within the family Poxviridae, which is the causative agent of Orf, a common epitheliotropic viral disease of sheep, goats, wild ruminants, and humans. In the present study, we sequenced the complete genomic sequences of two ORFV strains (ORFV-SY17, isolated from sheep, and ORFV-NA17, isolated from goat) and conducted the comparative analysis of multiple ORFVs. The complete genomic sequence of ORFV-SY17 was at length of 140,413bp, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 4267bp at both ends. The ORFV-NA17 strain displayed the similar genome structure with ORFV-SY17. The whole genomic sequence of ORFV-NA17 strain was 139,287bp in length and contained 132 ORFs flanked by ITRs of 3974bp. The overall G+C contents of ORFV-SY17 and ORFV-NA17 genome sequences were about 63.8% and 63.7%, respectively. The ITR sequences analysis showed that ORFV-SY17 and ORFV-NA17 contained the terminal BamHI sites and conserved telomere resolution sequences at both ends of their genome. In addition, comparative analysis of ORFs among ORFV-SY17, ORFV-NA17, and other ORFV strains revealed several sequence variations caused by insertions or deletions, especially in ORFs 005 and 116, which were very likely associated with host species. Phylogenetic analysis based on the complete genome sequences revealed that ORFV-SY17 was genetically closely related to NA1/11 and HN3/12 strains derived from sheep, while ORFV-NA17 was closely related to YX strain derived from goat. The multiple alignment of deduced amino acid sequences further revealed the genetic relationship between host species and genetic variations of ORFV strains. Taken together, the availability of genomic sequences of ORFV-SY17 and ORFV-NA17 strains from Jilin Province will aid in our understanding of the genetic diversity and evolution of ORFV strains in this region and can assist in distinguishing between ORFV strains that originate in sheep and goats.

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution.

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.

Spring loading a pre-cleavage intermediate for hairpin telomere formation.

The Borrelia telomere resolvase, ResT, forms the unusual hairpin telomeres of the linear Borrelia replicons in a process referred to as telomere resolution. Telomere resolution is a DNA cleavage and rejoining reaction that proceeds from a replicated telomere intermediate in a reaction with mechanistic similarities to that catalyzed by type IB topoisomerases. Previous reports have implicated the hairpin-binding module, at the end of the N-terminal domain of ResT, in distorting the DNA between the scissile phosphates so as to promote DNA cleavage and hairpin formation by the catalytic domain. We report that unwinding the DNA between the scissile phosphates, prior to DNA cleavage, is a key cold-sensitive step in telomere resolution. Through the analysis of ResT mutants, rescued by substrate modifications that mimic DNA unwinding between the cleavage sites, we show that formation and/or stabilization of an underwound pre-cleavage intermediate depends upon cooperation of the hairpin-binding module and catalytic domain. The phenotype of the mutants argues that the pre-cleavage intermediate promotes strand ejection to favor the forward reaction and that subsequent hairpin capture is a reversible reaction step. These reaction features are proposed to promote hairpin formation over strand resealing while allowing reversal back to substrate of aborted reactions.

Hairpin Telomere Resolvases

Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.

Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells.

Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. Using live-cell imaging, we show that expression of TRF1 or yellow fluorescent protein (YFP)-TRF1 fusion protein above endogenous levels prevents proper telomere resolution during mitosis. TRF1 overexpression results in telomere anaphase bridges and aggregates containing TRF1 protein and telomeric DNA. Site-specific protein cleavage of YFP-TRF1 by tobacco etch virus protease resolves telomere aggregates, indicating that telomere associations are mediated by TRF1. This study provides novel insight into the formation and resolution of telomere associations.

Construction and characterization of a Borrelia burgdorferi strain with conditional expression of the essential telomere resolvase, ResT.

Borrelia species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively in vitro, the essential nature of the resT gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a B. burgdorferi strain that carries an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible resT gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ~14,000 monomers per cell, similar to the ~15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT in vitro revealed the presence of replicated telomeres expected in replication intermediates.

The telomere resolvase of the Lyme disease spirochete, Borrelia burgdorferi, promotes DNA single-strand annealing and strand exchange

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpin telomeres. Hairpin telomeres present an uninterrupted DNA chain to the replication machinery overcoming the ‘end-replication problem’ for the linear replicons. Hairpin telomeres are formed from inverted repeat replicated telomere junctions by the telomere resolvase, ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. We report here that ResT also possesses single-strand annealing activity and a limited ability to promote DNA strand exchange reactions on partial duplex substrates. This combination of activities suggests ResT is a nexus between the seemingly distinct processes of telomere resolution and homologous recombination. Implications for hairpin telomere replication and linear plasmid recombination, including antigenic variation, are discussed.

Post-translational modifications of TRF1 and TRF2 and their roles in telomere maintenance

Telomeres, heterochromatic structures, found at the ends of linear eukaryotic chromosomes, function to protect natural chromosome ends from nucleolytic attack. Human telomeric DNA is bound by a telomere-specific six-subunit protein complex, termed shelterin/telosome. The shelterin subunits TRF1 and TRF2 bind in a sequence-specific manner to double-stranded telomeric DNA, providing a vital platform for recruitment of additional shelterin proteins as well as non-shelterin factors crucial for the maintenance of telomere length and structure. Both TRF1 and TRF2 are engaged in multiple roles at telomeres including telomere protection, telomere replication, sister telomere resolution and telomere length maintenance. Regulation of TRF1 and TRF2 in these various processes is controlled by post-translational modifications, at times in a cell-cycle-dependent manner, affecting key functions such as DNA binding, dimerization, localization, degradation and interactions with other proteins. Here we review the post-translational modifications of TRF1 and TRF2 and discuss the mechanisms by which these modifications contribute to the function of these two proteins.

Getting in (and out of) the loop: regulating higher order telomere structures

The DNA at the ends of linear chromosomes (the telomere) folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S-phase. Therefore, the coordinated regulation of telomere loop formation, maintenance, and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor known to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following “perspective” we outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

Herpes Simplex Virus Requires Poly(ADP-Ribose) Polymerase Activity for Efficient Replication and Induces Extracellular Signal-Related Kinase-Dependent Phosphorylation and ICP0-Dependent Nuclear Localization of Tankyrase 1

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) which localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Poly(ADP-ribosyl)ation of the nuclear mitotic apparatus (NuMA) protein by tankyrase 1 during mitosis is essential for sister telomere resolution and mitotic spindle pole formation. In interphase cells, tankyrase 1 resides in the cytoplasm, and its role therein is not well understood. In this study, we found that herpes simplex virus (HSV) infection induced extensive modification of tankyrase 1 but not tankyrase 2. This modification was dependent on extracellular signal-regulated kinase (ERK) activity triggered by HSV infection. Following HSV-1 infection, tankyrase 1 was recruited to the nucleus. In the early phase of infection, tankyrase 1 colocalized with ICP0 and thereafter localized within the HSV replication compartment, which was blocked in cells infected with the HSV-1 ICP0-null mutant R7910. In the absence of infection, ICP0 interacted with tankyrase 1 and efficiently promoted its nuclear localization. HSV did not replicate efficiently in cells depleted of both tankyrases 1 and 2. Moreover, XAV939, an inhibitor of tankyrase PARP activity, decreased viral titers to 2 to 5% of control values. We concluded that HSV targets tankyrase 1 in an ICP0- and ERK-dependent manner to facilitate its replication.

Cyclin B-dependent kinase 1 regulates human TRF1 to modulate the resolution of sister telomeres

Cyclin B-Cdk1 is a key mediator of mitotic entry, however little is known about its role in the separation of sister chromatids. Here we report that upon mitotic entry, Cdk1 specifically phosphorylates threonine 371 of TRF1, a telomere binding protein implicated in the regulation of sister telomere cohesion. Such phosphorylation is removed in late mitosis when Cdk1 activity is inhibited, indicative of a tight regulation of T371 phosphorylation. We show that T371 phosphorylation by Cdk1 keeps TRF1 free of chromatin and this phosphorylation is associated with loss of telomere-bound TRF1 and TIN2, and a reduction in telomere heterochromatin. We find that a phosphomimic mutation at T371 of TRF1 induces telomere deprotection, resulting in telomere loss and the formation of telomere fusions whereas a non-phosphorylatable substitution of T371 blocks sister telomere resolution, promotes micronuclei formation and impairs cell proliferation. Our work suggests that Cdk1 controls TRF1 association with telomeres to facilitate temporal telomere de-protection essential for sister telomere resolution.