Structural and biochemical characterisation of the human telomerase catalytic cycle and a telomerase dimer

Abstract

Telomeres are the regions of DNA at the ends of linear chromosomes. They are composed of repetitive G-rich sequences, GGTTAG in humans. An inherent problem for telomeres is their gradual shortening with every cell division, known as the end replication problem (Hayflick, 1965; Levy et al., 1992). Telomerase is the enzyme that counteracts this shortening by processively adding telomeric repeat sequences to the 3′ end of the DNA. Telomerase activity requires the telomerase reverse transcriptase subunit (TERT) and telomerase RNA (hTR in humans), which provides a template for DNA synthesis (Weinrich et al., 1997). Telomerase processivity is essential for telomere length maintenance since defects in processivity have been linked to disease (Armanios, 2022; Zaug et al., 2013). To synthesise a full telomeric repeat, telomerase must complete a highly complex and dynamic catalytic cycle. The mechanism and structural basis of how telomerase achieves processive telomeric repeat synthesis is not fully understood. High-resolution structures of human telomerase showed that it is monomeric for TERT and hTR and confirmed the composition of the enzyme (Ghanim et al., 2021; Liu et al., 2022; Wan et al., 2021). Telomerase has a dumbbell-like architecture with two lobes. The first lobe is referred to as the catalytic core because it contains TERT and is where DNA synthesis takes place. The second lobe contains two tetramers of H/ACA proteins and is therefore called the H/ACA lobe. These proteins play a key role in telomerase biogenesis and assembly. The H/ACA lobe also contains telomerase Cajal body protein 1 (TCAB1) that localises telomerase to Cajal bodies. The composition of the human telomerase enzyme has been a controversial topic within the telomere field. Initial size fractionation experiments suggested telomerase was approximately 600-650 kDa in size (Cohen et al., 2007; Wenz et al., 2001). Along with a low-resolution negative stain electron microscopy reconstruction (30 Å) (Sauerwald et al., 2013), these results lead to the first structural interpretation of human telomerase as a dimer of TERT, hTR, and dyskerin. Co-localisation experiments showed a sub-population of telomerase particles were dimeric for the TERT protein (Sauerwald et al., 2013; Wu et al., 2015). However, the high-resolution structure of human telomerase showed that the enzyme is typically monomeric (Ghanim et al., 2021). Therefore, it is still to be determined whether a dimeric form of telomerase exists can can be visualised. Chapter 2 of my PhD thesis presents a newly optimised purification method to reconstitute human telomerase complexes. This provided a platform for the following chapters. Chapter 3 reports structures of human telomerase in the initiation, elongation, and pre-termination stages of its catalytic cycle. I identified new motifs within the TERT protein that are critical for telomerase activity and showed how regions of hTR either side of the template region change conformation through the catalytic cycle. This allowed me to present an updated view of the telomerase catalytic cycle. Finally, in chapter 4 I show the first structure of a telomerase dimer. Unlike the previously reported dimer, this 1.2 MDa complex contains two complete copies of the telomerase holoenzyme and adopts an X shaped conformation. Dimerisation is mediated by hTR in the H/ACA lobes of the two telomerase particles. Mutagenesis experiments disrupting the dimer interface suggest a role for the dimer in the telomerase assembly pathway.