Protein interactions on telomeric retrotransposons in Drosophila

Telomere length in Drosophila is maintained by targeted transposition of three non-LTR retrotransposons: HeT-A, TART and TAHRE (HTT), but understanding the regulation of this process is hindered by our poor knowledge of HTT associated proteins. We have identified new protein components of the HTT array: Chromator (Chro), the TRF2/DREF complex and the sumoylation machinery. Chro was localized on telomeric HTT arrays by immunostaining, where it may interact with Prod directly, as indicated by yeast two-hybrid interaction, co-IP, and colocalization on polytene chromosomes. The TRF2/DREF complex may promote the open structure of HTT chromatin. The protein interactions controlling HTT chromatin structure and telomere length may be modulated by sumoylation.     

Evidence for genomic regulation of the telomeric activity in Drosophila melanogaster

The structural integrity of TART elements has been used as reporter of instability at chromosomal ends in numerous Drosophila stocks and over time in an unstable stock. The results show that telomeric activity is a regulated process that may differ between the stocks as well as over time within a stock. This revised version was published online in July 2006 with corrections to the Cover Date.     

The role of HeT-A and TART retrotransposons in Drosophila telomere capping

Drosophila telomeres contain multiple copies of HeT-A and TART retrotransposons. These elements specifically transpose to chromosomal ends, compensating for loss of terminal nucleotides that occurs at each cycle of DNA replication. We have investigated the role of these sequences in the formation of telomere–telomere attachments induced by mutations in the UbcD1 gene. We have constructed UbcD1 mutant males carrying terminally deleted X chromosomes devoid of both HeT-A and TART sequences. Cytological analysis of larval neuroblasts from these males revealed that telomeres lacking HeT-A and TART and normal telomeres that contain these sequences participate in telomeric fusions with comparable frequencies. These results indicate that the UbcD1 substrate(s) binds chromosomal termini in a sequence-independent manner. Previous studies have shown that the telomere-capping protein HP1 also binds telomeres lacking HeT-A and TART. Taken together, these findings strongly suggest that the assembly of DNA–protein complexes that protect chromosome ends from fusions do not require specific terminal sequences.     

Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons

Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.     

MTV sings jubilation for telomere biology in Drosophila

Telomere protects the ends of linear chromosomes. Telomere dysfunction fuels genome instability that can lead to diseases such as cancer. For over 30 years, Drosophila has fascinated the field as the only major model organism that does not rely on the conserved telomerase enzyme for end protection. Instead of short DNA repeats at chromosome ends, Drosophila has domesticated retrotransposons. In addition, telomere protection can be entirely sequence-independent under normal laboratory conditions, again dissimilar to what has been established for telomerase-maintained systems. Despite these major differences, recent studies from us and others have revealed remarkable similarities between the 2 systems. In particular, with the identification of the MTV complex as an ssDNA binding complex essential for telomere integrity in Drosophila (Zhang et al. 2016 Plos Genetics), we have now established several universal principles that are intrinsic to chromosome extremities but independent of the underlying DNA sequences or the telomerase enzyme. Telomere studies in Drosophila will continue to yield fundamental insights that are instrumental to the understanding of the evolution of telomere and telomeric functions.     

Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

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Timeless preserves telomere length by promoting efficient DNA replication through human telomeres

A variety of telomere protection programs are utilized to preserve telomere structure. However, the complex nature of telomere maintenance remains elusive. The Timeless protein associates with the replication fork and is thought to support efficient progression of the replication fork through natural impediments, including replication fork block sites. However, the mechanism by which Timeless regulates such genomic regions is not understood. Here, we report the role of Timeless in telomere length maintenance. We demonstrate that Timeless depletion leads to telomere shortening in human cells. This length maintenance is independent of telomerase, and Timeless depletion causes increased levels of DNA damage, leading to telomere aberrations. We also show that Timeless is associated with Shelterin components TRF1 and TRF2. Timeless depletion slows telomere replication in vitro, and Timeless-depleted cells fail to maintain TRF1-mediated accumulation of replisome components at telomeric regions. Furthermore, telomere replication undergoes a dramatic delay in Timeless-depleted cells. These results suggest that Timeless functions together with TRF1 to prevent fork collapse at telomere repeat DNA and ensure stable maintenance of telomere length and integrity.     

Timeless preserves telomere length by promoting efficient DNA replication through human telomeres

A variety of telomere protection programs are utilized to preserve telomere structure. However, the complex nature of telomere maintenance remains elusive. The Timeless protein associates with the replication fork and is thought to support efficient progression of the replication fork through natural impediments, including replication fork block sites. However, the mechanism by which Timeless regulates such genomic regions is not understood. Here, we report the role of Timeless in telomere length maintenance. We demonstrate that Timeless depletion leads to telomere shortening in human cells. This length maintenance is independent of telomerase, and Timeless depletion causes increased levels of DNA damage, leading to telomere aberrations. We also show that Timeless is associated with Shelterin components TRF1 and TRF2. Timeless depletion slows telomere replication in vitro, and Timeless-depleted cells fail to maintain TRF1-mediated accumulation of replisome components at telomeric regions. Furthermore, telomere replication undergoes a dramatic delay in Timeless-depleted cells. These results suggest that Timeless functions together with TRF1 to prevent fork collapse at telomere repeat DNA and ensure stable maintenance of telomere length and integrity.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

piggyBac转座子AgoPLE1.1在黑腹果蝇种系转化中的应用

[目的]通过检测黑腹果蝇 DDrosophiila melanogaster中piggyBac(PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力.[方法]构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体...     

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent‐strand DNA

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1‐STN1‐TEN1) proteins, which form a single‐stranded DNA‐binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent‐strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5’ DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA‐binding ability of CST is required for blocking MRE11‐mediated nascent‐strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent‐strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.     

Initiation of DNA replication in eukaryotes is an intriguing cascade of protein interactions

Initiation of eukaryotic DNA replication is a complex process including the recognition of initiation sites on DNA, multi-step DNA preparation for duplication, and assembly of multi-protein complexes capable of beginning DNA synthesis at initiation sites. The process starts at the late M phase and lasts till the appropriate time of the S phase for each initiation site. A chain of interesting interactions between Orc1p-6p, Cdc6p, Mcm2p-7p, Mcm10p, Cdt1, Cdc45p, Dbf4/Cdc7p, RPA, and DNA polymerase takes place during this period. The sequence of these interactions is controlled by cyclin-dependent kinases, as well as by ubiquitin-dependent proteolysis in the proteasome. This review summarizes the data on proteins initiating DNA replication and factors controlling their activities.     

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera,Belostoma)

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome–autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elegans (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. elongatum (2n = 26 + X₁X₂Y/X₁X₁X₂X₂), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG)_n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere–telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.     

Structure,dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD⁺ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.