Uncapping and deregulation of telomeres lead to detrimental cellular consequences in yeast

Telomeres are the protein-nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen "monster" cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.     

Function, replication and structure of the mammalian telomere

Telomeres are specialized structures at the ends of linear chromosomes that were originally defined functionally based on observations first by Muller (1938) and subsequently by McClintock (1941) that naturally occurring chromosome ends do not behave as double-stranded DNA breaks, in spite of the fact that they are the physical end of a linear, duplex DNA molecule. Double-stranded DNA breaks are highly unstable entities, being susceptible to nucleolytic attack and giving rise to chromosome rearrangements through end-to-end fusions and recombination events. In contrast, telomeres confer stability upon chromosome termini, as evidenced by the fact that chromosomes are extraordinarily stable through multiple cell divisions and even across evolutionary time. This protective function of telomeres is due to the formation of a nucleoprotein complex that sequesters the end of the DNA molecule, rendering it inaccessible to nucleases and recombinases as well as preventing the telomere from activating the DNA damage checkpoint pathways. The capacity of a functional end-protective complex to form is dependent upon maintenance of sufficient telomeric DNA. We have learned a great deal about telomere structure and how this specialized nucleoprotein complex confers stability on chromosome ends since the original observations that defined telomeres were made. This review summarizes our current understanding of mammalian telomere replication, structure and function.     

Defending the end zone: Studying the players involved in protecting chromosome ends

The linear nature of eukaryotic chromosomes leaves natural DNA ends susceptible to triggering DNA damage responses. Telomeres are specialized nucleoprotein structures that comprise the “end zone” of chromosomes. Besides having specialized sequences and structures, there are six resident proteins at telomeres that play prominent roles in protecting chromosome ends. In this review, we discuss this team of proteins, termed shelterin, and how it is involved in regulating DNA damage signaling, repair and replication at telomeres.     

Give me a break: How telomeres suppress the DNA damage response

Linear organization of the genome requires mechanisms to protect and replicate chromosome ends. To this end eukaryotic cells evolved telomeres, specialized nucleoproteic complexes, and telomerase, the enzyme that maintains the telomeric DNA. Telomeres allow cells to distinguish chromosome ends from sites of DNA damage. In mammalian cells this is accomplished by a protein complex, termed shelterin, that binds to telomeric DNA and is able to shield chromosome ends from the DNA damage machinery. In recent years, we have seen major advances in our understanding of how this protein complex works due to the generation of mouse models carrying mutations of individual shelterin components. This review will focus on our current understanding of how the shelterin complex is able to suppress the DNA damage response pathways, and on the cellular and organismal outcomes of telomere dysfunction.     

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Thermodynamic and electrostatic properties of ternary Oxytricha nova TEBP-DNA complex

Telomeres constitute the nucleoprotein ends of eukaryotic chromosomes which are essential for their proper function. Telomere end binding protein (TEBP) from Oxytricha nova was among the first telomeric proteins, which were well characterized biologically. TEBP consists of two protein subunits (alpha, beta) and forms a ternary complex with single stranded telomeric DNA containing tandem repeats TTTTGGGG. This work presents the characterization of the thermodynamic and electrostatic properties of this complex by computational chemistry methods (continuum Poisson-Boltzmann and solvent accessible surface calculations). Our calculations give a new insight into molecular properties of studied system. Based on the thermodynamic analysis we provide a rationale for the experimental observation that alpha and ssDNA forms a binary complex and the beta subunit joins alpha:ssDNA complex only after the latter is formed. Calculations of distribution of the molecular electrostatic potential for protein subunits alone and for all possible binary complexes revealed the important role of the "guiding funnel" potential generated by alpha:ssDNA complex. This potential may help the beta subunit to dock to the already formed alpha:DNA intermediate in highly steric and electrostatic favorable manner. Our pK(a) calculations of TEBP are able to explain the experimental mobility shifts of the complex in electrophoretic non-denaturating gels.     

Cell-cycle-dependent telomere elongation by telomerase in budding yeast

Telomeres are essential for the stability and complete replication of linear chromosomes. Telomere elongation by telomerase counteracts the telomere shortening due to the incomplete replication of chromosome ends by DNA polymerase. Telomere elongation is cell-cycle-regulated and coupled to DNA replication during S-phase. However, the molecular mechanisms that underlie such cell-cycle-dependent telomere elongation by telomerase remain largely unknown. Several aspects of telomere replication in budding yeast, including the modulation of telomere chromatin structure, telomere end processing, recruitment of telomere-binding proteins and telomerase complex to telomere as well as the coupling of DNA replication to telomere elongation during cell cycle progression will be discussed, and the potential roles of Cdk (cyclin-dependent kinase) in these processes will be illustrated.     

Brh2 and Rad51 promote telomere maintenance in Ustilago maydis,a new model system of DNA repair proteins at telomeres

Recent studies implicate a number of DNA repair proteins in mammalian telomere maintenance. However, because several key repair proteins in mammals are missing from the well-studied budding and fission yeast, their roles at telomeres cannot be modeled in standard fungi. In this report, we explored the dimorphic fungus Ustilago maydis as an alternative model for telomere research. This fungus, which belongs to the phylum Basidiomycota, has a telomere repeat unit that is identical to the mammalian repeat, as well as a constellation of DNA repair proteins that more closely mimic the mammalian collection. We showed that the two core components of homology-directed repair (HDR) in U. maydis, namely Brh2 and Rad51, both promote telomere maintenance in telomerase positive cells, just like in mammals. In addition, we found that Brh2 is localized to telomeres in vivo, suggesting that it acts directly at chromosome ends. We surveyed a series of mutants with DNA repair defects, and found many of them to have short telomeres. Our results indicate that factors involved in DNA repair are probably also needed for optimal telomere maintenance in U. maydis, and that this fungus is a useful alternative model system for telomere research.     

The preference for a 3' homologous end is intrinsic to RecA-promoted strand exchange

The recA protein (RecA) promotes DNA pairing and strand exchange optimally in the presence of single-stranded binding protein (SSB). Under these conditions, 3' homologous ends are essential for stable joint molecule formation between linear single-stranded DNA (ssDNA) and supercoiled DNA (i.e. 3' ends are 50-60 times more reactive than 5' ends). Linear ssDNAs with homology at the 5' end do not participate in pairing. In the absence of SSB, the strand exchange reaction is less efficient; however, linear ssDNAs with 3' end homology are still 5- to 10-fold more reactive than those with 5' end homology. The preference for a 3' homologous end in the absence of SSB suggests that this is an intrinsic property of RecA-promoted strand exchange. The preferential reactivity of 3' homologous ends is likely to be a consequence of the polarity of polymerization of RecA on ssDNA. Specifically, since RecA polymerizes in the 5'----3' direction, 3' ends are more likely to be coated with RecA and, hence, will be more reactive than 5' ends.     

Pathways connecting telomeres and p53 in senescence, apoptosis, and cancer

The ends of eukaryotic chromosomes are protected by specialized structures termed telomeres that serve in part to prevent the chromosome end from activating a DNA damage response. However, this important function for telomeres in chromosome end protection can be lost as telomeres shorten with cell division in culture or in self-renewing tissues with advancing age. Impaired telomere function leads to induction of a DNA damage response and activation of the tumor suppressor protein p53. p53 serves a critical role in enforcing both senescence and apoptotic responses to dysfunctional telomeres. Loss of p53 creates a permissive environment in which critically short telomeres are inappropriately joined to generate chromosomal end-to-end fusions. These fused chromosomes result in cycles of chromosome fusion-bridge-breakage, which can fuel cancer initiation, especially in epithelial tissues, by facilitating changes in gene copy number.     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres

Telomeres protect chromosome ends from being detected as lesions and from triggering DNA damage checkpoints. Paradoxically, telomere function depends on checkpoint proteins such as ATM and ATR, but a molecular model explaining this seemingly contradictory relationship has been missing so far. Here we show that the DNA damage machinery acts on telomeres in at least two independent steps. First, the ATR-dependent machinery is recruited to telomeres before telomere replication is completed, likely in response to single-stranded DNA resulting from replication fork stalling. Second, after replication, telomeres attract ATM and the homologous recombination (HR) machinery. In vivo and in vitro results suggest that the HR machinery is required for formation of a telomere-specific structure at chromosome ends after replication. Our results suggest that telomere ends need to be recognized as DNA damage to complete end replication and to acquire a structure that is essential for function.     

Retroviruses as mutagens: Insertion and excision of a nontransforming provirus alter expression of a resident transforming provirus

Integration of retroviral DNA appears to occur randomly in host genomes, suggesting that retroviruses can act as insertion mutagens. We have confirmed this prediction by showing that the nontransforming retrovirus, Moloney murine leukemia virus (M-MuLV), can insert its provirus within the selectable target provided by a single provirus in a clonal rat cell line (B31) transformed by Rous sarcoma virus (RSV). Analysis of over 60 morphological revertants of M-MuLV-superinfected B31 cells revealed two lines with inserts of M-MuLV proviruses within the RSV provirus but outside the transforming gene of RSV (src), at sites 0.6 and 4.0 kb from the 5′ end. The inserts did not inactivate initiation of RSV RNA synthesis but did affect elongation or processing, or both, generating species with the 5′ end of RSV RNA linked to sequences that presumably derive from the inserted M-MuLV DNA. In one mutant line, most of the insert was excised at low frequency, apparently by homologous recombination between repeated sequences at the ends of M-MuLV DNA. After excision, RSV src mRNA was present in normal amounts, and the cells resumed a transformed appearance. In at least four independent lines, large portions of the left end of the RSV provirus (from 1 to 6 kb) and variable amounts of leftward flanking cellular DNA (from 0.5 to 10–15 kb or more) were deleted, without nearby insertions of M-MuLV DNA. The deletions removed the putative promoter for synthesis of RSV RNA; in the two cases examined, no RSV RNA was detected. These deletions may represent a second mutational effect of the superinfection by M-MuLV.