Activity of zebrafish THAP9 transposase and zebrafish P element-like transposons.

Transposable elements are mobile DNA segments that are found ubiquitously across the three domains of life. One family of transposons, called P elements, were discovered in the fruit fly Drosophila melanogaster. Since their discovery, P element transposase-homologous genes (called THAP-domain containing 9 or THAP9) have been discovered in other animal genomes. Here, we show that the zebrafish (Danio rerio) genome contains both an active THAP9 transposase (zfTHAP9) and mobile P-like transposable elements (called Pdre). zfTHAP9 transposase can excise one of its own elements (Pdre2) and Drosophila P elements. Drosophila P element transposase (DmTNP) is also able to excise the zebrafish Pdre2 element, even though it's distinct from the Drosophila P element. However, zfTHAP9 cannot transpose Pdre2 or Drosophila P elements, indicating partial transposase activity. Characterization of the N-terminal THAP DNA binding domain of zfTHAP9 shows distinct DNA binding site preferences from DmTNP and mutation of the zfTHAP9, based on known mutations in DmTNP, generated a hyperactive protein,. These results define an active vertebrate THAP9 transposase that can act on the endogenous zebrafish Pdre and Drosophila P elements.

2.7 Å cryo-EM structure of human telomerase H/ACA ribonucleoprotein.

Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase is comprised of two distinct functional lobes tethered by telomerase RNA (hTR): a catalytic core, responsible for DNA extension; and a Hinge and ACA (H/ACA) box RNP, responsible for telomerase biogenesis. H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs, which is critical for the biogenesis of the spliceosome and ribosome. Much of our structural understanding of eukaryotic H/ACA RNPs comes from structures of the human telomerase H/ACA RNP. Here we report a 2.7 Å cryo-electron microscopy structure of the telomerase H/ACA RNP. The significant improvement in resolution over previous 3.3 Å to 8.2 Å structures allows us to uncover new molecular interactions within the H/ACA RNP. Many disease mutations are mapped to these interaction sites. The structure also reveals unprecedented insights into a region critical for pseudouridylation in canonical H/ACA RNPs. Together, our work advances understanding of telomerase-related disease mutations and the mechanism of pseudouridylation by eukaryotic H/ACA RNPs.

Structural basis of telomeric nucleosome recognition by shelterin factor TRF1.

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1. Our structures reveal that TRF1 binds unwrapped nucleosomal DNA ends by engaging both the nucleosomal DNA and the histone octamer. Unexpectedly, TRF1 binding shifts the register of the nucleosomal DNA by 1 bp. We discovered that phosphorylation of the TRF1 C terminus and a noncanomical DNA binding surface on TRF1 are critical for its association with telomeric nucleosomes. These insights into shelterin-chromatin interactions have crucial implications for understanding telomeric chromatin organization and other roles of shelterin at telomeres including replication and transcription.

Structural basis of human telomerase recruitment by TPP1-POT1.

Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

Structure of human telomerase holoenzyme with bound telomeric DNA.

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication1. In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease2. A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture3, comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.

Mechanism and regulation of P element transposition.

P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans. This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.

Structure of a P element transposase-DNA complex reveals unusual DNA structures and GTP-DNA contacts.

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.