Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization.

The regulation of telomere length in mammals is crucial for chromosome end-capping and thus for maintaining genome stability and cellular lifespan. This process requires coordination between telomeric protein complexes and the ribonucleoprotein telomerase, which extends the telomeric DNA. Telomeric proteins modulate telomere architecture, recruit telomerase to accessible telomeres and orchestrate the conversion of the newly synthesized telomeric single-stranded DNA tail into double-stranded DNA. Dysfunctional telomere maintenance leads to telomere shortening, which causes human diseases including bone marrow failure, premature ageing and cancer. Recent studies provide new insights into telomerase-related interactions (the 'telomere replisome') and reveal new challenges for future telomere structural biology endeavours owing to the dynamic nature of telomere architecture and the great number of structures that telomeres form. In this Review, we discuss recently determined structures of the shelterin and CTC1-STN1-TEN1 (CST) complexes, how they may participate in the regulation of telomere replication and chromosome end-capping, and how disease-causing mutations in their encoding genes may affect specific functions. Major outstanding questions in the field include how all of the telomere components assemble relative to each other and how the switching between different telomere structures is achieved.

Structures of the human CST-Polα-primase complex bound to telomere templates.

The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.

Models for human telomere C-strand fill-in by CST-Polα-primase.

Telomere maintenance is essential for the genome integrity of eukaryotes, and this function is underpinned by the two-step telomeric DNA synthesis process: telomere G-overhang extension by telomerase and complementary strand fill-in by DNA polymerase alpha-primase (polα-primase). Compared to the telomerase step, the telomere C-strand fill-in mechanism is less understood. Recent studies have provided new insights into how telomeric single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) and polα-primase coordinate to synthesize the telomeric C-strand for telomere overhang fill-in. Cryogenic electron microscopy (cryo-EM) structures of CST-polα-primase complexes have provided additional insights into how they assemble at telomeric templates and de novo synthesize the telomere C-strand. In this review, we discuss how these latest findings coalesce with existing understanding to develop a human telomere C-strand fill-in mechanism model.

Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.

CST does not evict elongating telomerase but prevents initiation by ssDNA binding.

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties.

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.

Dynamics and stability of polymorphic human telomeric G-quadruplex under tension.

As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K(+) concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability. Our results show that the kinetically favored folding pathway is through a short-lived intermediate state to a longer-lived state. By examining the force dependence of transition rates, the force-dependent transition free energy landscape for this pathway is determined. In addition, an ultra-long-lived form of telomeric G4 structure with a much stronger mechanical stability is identified.

The nucleoid-associated protein Dan organizes chromosomal DNA through rigid nucleoprotein filament formation in E. coli during anoxia.

Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan-DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation.

Mechanism of DNA organization by Mycobacterium tuberculosis protein Lsr2.

Bacterial nucleoid-associated proteins, such as H-NS-like proteins in Enterobacteriaceae, are abundant DNA-binding proteins that function in chromosomal DNA organization and gene transcription regulation. The Mycobacterium tuberculosis Lsr2 protein has been proposed to be the first identified H-NS analogue in Gram-positive bacteria based on its capability to complement numerous in vivo functions of H-NS. Here, we report that Lsr2 cooperatively binds to DNA forming a rigid Lsr2 nucleoprotein complex that restricts DNA accessibility, similar to H-NS. On large DNA, the rigid Lsr2 nucleoprotein complexes can mediate DNA condensation into highly compact DNA conformations. In addition, the responses of Lsr2 nucleoprotein complex to environmental factors (salt concentration, temperature and pH) were studied over physiological ranges. These results provide mechanistic insights into how Lsr2 may mediate its gene silencing, genomic DNA protection and organization functions in vivo. Finally, our results strongly support that Lsr2 is an H-NS-like protein in Gram-positive bacteria from a structural perspective.

Gene silencing H-NS paralogue StpA forms a rigid protein filament along DNA that blocks DNA accessibility.

Nucleoid-associated proteins are bacterial proteins that are responsible for chromosomal DNA compaction and global gene regulation. One such protein is Escherichia coli Histone-like nucleoid structuring protein (H-NS) which functions as a global gene silencer. Whereas the DNA-binding mechanism of H-NS is well-characterized, its paralogue, StpA which is also able to silence genes is less understood. Here we show that StpA is similar to H-NS in that it is able to form a rigid filament along DNA. In contrast to H-NS, the StpA filament interacts with a naked DNA segment to cause DNA bridging which results in simultaneous stiffening and bridging of DNA. DNA accessibility is effectively blocked after the formation of StpA filament on DNA, suggesting rigid filament formation is the important step in promoting gene silencing. We also show that >1 mM magnesium promotes higher order DNA condensation, suggesting StpA may also play a role in chromosomal DNA packaging.

Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing.

Sample loss due to air-water interface (AWI) interactions is a significant challenge during cryo-electron microscopy (cryo-EM) sample grid plunge freezing. We report that small Late Embryogenesis Abundant (LEA) proteins, which naturally bind to AWI, can protect samples from AWI damage during plunge freezing. This protection is demonstrated with two LEA proteins from nematodes and tardigrades, which rescued the cryo-EM structural determination outcome of two fragile multisubunit protein complexes.

Structures of human primosome elongation complexes.

The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA-DNA primers.

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.

Reconstitution of human shelterin complexes reveals unexpected stoichiometry and dual pathways to enhance telomerase processivity.

The human shelterin proteins associate with telomeric DNA to confer telomere protection and length regulation. They are thought to form higher-order protein complexes for their functions, but studies of shelterin proteins have been mostly limited to pairs of proteins. Here we co-express various human shelterin proteins and find that they form defined multi-subunit complexes. A complex harboring both TRF2 and POT1 has the strongest binding affinity to telomeric DNA substrates comprised of double-stranded DNA with a 3' single-stranded extension. TRF2 interacts with TIN2 with an unexpected 2:1 stoichiometry in the context of shelterin (RAP12:TRF22:TIN21:TPP11:POT11). Tethering of TPP1 to the telomere either via TRF2-TIN2 or via POT1 gives equivalent enhancement of telomerase processivity. We also identify a peptide region from TPP1 that is both critical and sufficient for TIN2 interaction. Our findings reveal new information about the architecture of human shelterin and how it performs its functions at telomeres.

Regulation of Bacterial DNA Packaging in Early Stationary Phase by Competitive DNA Binding of Dps and IHF.

The bacterial nucleoid, a bacterial genome packed by nucleoid binding proteins, forms the physical basis for cellular processes such as gene transcription and DNA replication. Bacteria need to dynamically modulate their nucleoid structures at different growth phases and in response to environmental changes. At the nutrients deficient stationary phase, DNA-binding proteins from starved cells (Dps) and Integration host factors (IHF) are the two most abundant nucleoid associated proteins in E. coli. Yet, it remains unclear how the nucleoid architecture is controlled by the interplay between these two proteins, as well as the nucleoid's response to environmental changes. This question is addressed here using single DNA manipulation approach. Our results reveal that the two proteins are differentially selected for DNA binding, which can be tuned by changing environmental factors over physiological ranges including KCl (50-300 mM), MgCl2 (0-10 mM), pH (6.5-8.5) and temperature (23-37 °C). Increasing pH and MgCl2 concentrations switch from Dps-binding to IHF-binding. Stable Dps-DNA and IHF-DNA complexes are insensitive to temperature changes for the range tested. The environment dependent selection between IHF and Dps results in different physical organizations of DNA. Overall, our findings provide important insights into E. coli nucleoid architecture.

Complex kinetics of DNA condensation revealed through DNA twist tracing.

Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.

Nucleoprotein filament formation is the structural basis for bacterial protein H-NS gene silencing.

H-NS is an abundant nucleoid-associated protein in bacteria that globally silences genes, including horizontally-acquired genes related to pathogenesis. Although it has been shown that H-NS has multiple modes of DNA-binding, which mode is employed in gene silencing is still unclear. Here, we report that in H-NS mutants that are unable to silence genes, are unable to form a rigid H-NS nucleoprotein filament. These results indicate that the H-NS nucleoprotein filament is crucial for its gene silencing function, and serves as the fundamental structural basis for gene silencing by H-NS and likely other H-NS-like bacterial proteins.

Isolation and characterization of a phenol-degrading bacterium from an industrial activated sludge.

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.0% identity to 16S rRNA gene sequences of Acinetobacter calcoaceticus. Strain EDP3 could aerobically grow on a number of aromatic compounds, such as phenol, sodium benzoate, p-hydroxybenzoate, phenylacetate, benzene, ethylbenzene, benzylalcohol, and so on. In particular, it could mineralize up to 1,000 mg l(-1) phenol at room temperature (25 degrees C). The growth kinetics of strain EDP3 on phenol as a sole carbon and energy source at 25 degrees C can be described using the Haldane equation. It has a maximal specific growth rate (mu(max)) of 0.28 h(-1), a half-saturation constant (K(S)) of 1,167.1 mg l(-1), and a substrate inhibition constant (Ki) of 58.5 mg l(-1). Values of yield coefficient (Y(X/S)) are between 0.4 and 0.6 mg dry cell (mg phenol)(-1). Strain EDP3 has high tolerance to the toxicity of phenol (up to 1,000 mg l(-1)). It therefore could be an excellent candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated soils.