Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

Ku complex controls the replication time of DNA in telomere regions

We have investigated whether the Ku complex is involved in regulating DNA replication in the yeast Saccharomyces cerevisiae. We find that Ku proteins control the replication time of telomeric regions; replication origins located close to telomeres or within subtelomeric repeat sequences normally initiate late, but are activated much earlier in mutants lacking Ku function. In contrast, origins distant from telomeres initiate replication at the normal time. Ku is one of the first components identified as important for replication timing, and specification of the replication time of chromosome ends by Ku is consistent with its role in maintaining telomere localization.     

Cdc13 both positively and negatively regulates telomere replication

Cdc13 is a single-strand telomeric DNA-binding protein that positively regulates yeast telomere replication by recruiting telomerase to chromosome termini through a site on Cdc13 that is eliminated by the cdc13-2 mutation. Here we show that Cdc13 has a separate role in negative regulation of telomere replication, based on analysis of a new mutation, cdc13-5. Loss of this second regulatory activity results in extensive elongation of the G strand of the telomere by telomerase, accompanied by a reduced ability to coordinate synthesis of the C strand. Both the cdc13-5 mutation and DNA polymerase alpha mutations (which also exhibit elongated telomeres) are suppressed by increased expression of the Cdc13-interacting protein Stn1, indicating that Stn1 coordinates action of the lagging strand replication complex with the regulatory activity of CDC13. However, the association between Cdc13 and Stn1 is abolished by cdc13-2, the same mutation that eliminates the interaction between Cdc13 and telomerase. We propose that Cdc13 participates in two regulatory steps-first positive, then negative-as a result of successive binding of telomerase and the negative regulator Stn1 to overlapping sites on Cdc13. Thus, Cdc13 coordinates synthesis of both strands of the telomere by first recruiting telomerase and subsequently limiting G-strand synthesis by telomerase in response to C-strand replication.     

Normal human chromosomes have long G-rich telomeric overhangs at one end

Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200±75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3′ overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.     

Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication

Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.     

SUMO修饰与端粒维持

端粒(telomere)长度维持在一定的范围内是细胞正常生理功能的一个重要的基础,端粒长度的变化可导致两个截然相反的病理生理过程-癌变和衰老,端粒过长可引起细胞永生化而癌变,端粒进行性缩短则有丝分裂能力下降导致细胞衰老[1].端粒的长度和结构依赖端粒酶的活性及端粒蛋白复合体(shelterin)的调节[2],端粒酶的激活可引起端粒DNA序列增加而延长细胞的寿命.泛素样小分子修饰(small ubiquin-like modifier,SUMO修饰)在不同的端粒维持机制中的作用途径不同,SUMO修饰可激活端粒酶的活性,促进依赖端粒酶合成端粒的途径,SUMO修饰也可促进同源重组途径合成端粒的能力[3],增加端粒的长度,在保持端粒的长度上发挥重要的调节作用.     

Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends

Ku is a conserved DNA end-binding protein that plays various roles at different kinds of DNA ends. At telomeres, Ku is part of the structure that protects the chromosome end, whereas at broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway. Here, we present evidence of a new role for Ku that impacts both telomere-length maintenance and DNA repair in Saccharomyces cerevisiae. We show that Ku binds TLC1, the RNA component of telomerase. We also describe a novel separation-of-function allele of Ku that is specifically defective in TLC1 binding. In this mutant, telomeres are short and the kinetics of telomere addition are slow, but other Ku-dependent activities, such as chromosome end protection and NHEJ, are unaffected. At low frequency, yeast will use telomerase to heal DNA damage by capping the broken chromosome with telomeric DNA sequences. We show that when Ku's ability to bind TLC1 is disrupted, DNA repair via telomere healing is reduced 10- to 100-fold, and the spectrum of sequences that can acquire a telomere changes. Thus, the interaction between Ku and TLC1 RNA enables telomerase to act at both broken and normal chromosome ends.     

端粒/端粒酶在恶性血液病中的研究进展

端粒是真核细胞染色体末端特有的一段DNA序列和几种特异性蛋白质构成的复合物。端粒酶是一种核糖核酶,能以自身RNA为模板,合成端粒DNA,从而维持端粒的长度。端粒/端粒酶的表达调控对肿瘤的发生发展及细胞的衰老、永生化起着重要的作用,深入研究端粒/端粒酶的结构功能及其在恶性血液病细胞和正常造血细胞中的表达和调控,将有助于达到控制和治疗恶性血液病的目的。     

Kinetics of DNA replication and telomerase reaction at a single-seeded telomere in human cells

In most cancer cells, telomerase is activated to elongate telomere DNA, thereby ensuring numerous rounds of cell divisions. It is thus important to understand how telomerase and the replication fork react with telomeres in human cells. However, the highly polymorphic and repetitive nature of the nucleotide sequences in human subtelomeric regions hampers the precise analysis of sequential events taking place at telomeres in S phase. Here, we have established HeLa cells harboring a single-seeded telomere abutted by a unique subtelomere DNA sequence, which has enabled us to specifically focus on the seeded telomere. We have also developed a modified chromatin immunoprecipitation (ChIP) method that uses restriction digestion instead of sonication to fragment chromatin DNA (RES-ChIP), and a method for immunoprecipitating 5-bromo-2'-deoxyuridine (BrdU)-labeled single-stranded DNA by incubating DNA with anti-BrdU antibody in the nondenaturing condition. We have shown that DNA replication of the seeded telomere takes place during a relatively narrow time window in S phase, and telomerase synthesizes telomere DNA after the replication. Moreover, we have demonstrated that the telomerase catalytic subunit TERT associates with telomeres before telomere DNA replication. These results provide a temporal and spatial framework for understanding DNA replication and telomerase reaction at human telomeres.     

Pol12, the B subunit of DNA polymerase alpha, functions in both telomere capping and length regulation

The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase alpha and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.     

Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 A resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed.     

Relative telomere length and cognitive decline in the Nurses' Health Study

Telomeres are short DNA repeats on the ends of mammalian chromosomes, which can undergo incomplete replication leading to gradual shortening with each cell cycle. Age and oxidative stress are contributors to telomere shortening; thus, telomere length may be a composite measure of biologic aging, and a potential predictor of health status in older adults. We evaluated whether relative telomere length (the proportion of telomere repeat copy number to single gene copy number, using a real-time PCR method) predicts cognitive decline measured ten years later among ～ 2000 older participants in the Nurses' Health Study (NHS). Mixed linear regression was used to evaluate mean differences in cognitive decline according to telomere length. After adjustment for potential confounders, we found that decreasing telomere length was associated with more cognitive decline, although associations were modest (e.g. for a global score, averaging all six tests in our cognitive battery, mean difference=0.03 standard units per SD increase in telomere length; p=0.04). The magnitude of these estimates was similar to the differences we find in this cohort for women one year apart in age (e.g. the differences that we observe between women who are 73 versus 74 years of age); thus, our results suggest that telomere length is not a particularly powerful marker of impending cognitive decline.     

Molecular basis of telomere syndrome caused by CTC1 mutations

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase α-primase (polα-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA polα-primase to promote faithful telomere DNA replication.     

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.     

RNA primer removal and gap filling on a model minicircle replication intermediate

Replication of kinetoplast DNA minicircles in Crithidia fasciculata occurs by a unidirectional mechanism involving continuous synthesis of one strand (L strand) and discontinuous synthesis of the complementary strand (H strand). L-strands are initiated by RNA priming at alternate origins (A and B) resulting in daughter molecules with a single nick or gap in the L strand at either ori A or ori B. Some of the gapped molecules contain ribonucleotides at the 5' side of the gap. We have investigated the ability of recombinant forms of kinetoplast replication proteins, DNA polymerase beta and structure specific endonuclease 1, to repair gaps in a model minicircle substrate. Structure specific endonuclease 1 was shown to efficiently remove all ribonucleotides from the 5' side of the model substrate by stepwise cleavage of the RNA primer. Polymerase beta was then able to extend the 3' terminus of the gap to yield a nicked molecule capable of covalent joining by a DNA ligase. These results demonstrate that the nuclease and polymerase enzymes present at antipodal protein complexes flanking the kinetoplast disk are capable of complete RNA primer removal and subsequent gap filling of newly synthesized minicircle L strands.     

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres.     

Ku acts in a unique way at the mammalian telomere to prevent end joining

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.