A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

Abstract

1Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, 2 preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have 3 previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended 4 by human and ciliate telomerase. However, a mechanistic understanding of the interaction of 5 telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence 6 resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a 7 mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated 8 by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then 9 proceeds to the end of the RNA template. It is only through the large conformational change 10 of translocation following synthesis that the G-quadruplex structure is completely unfolded to 11 a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or 12 by chemical modification does not always inhibit G4 unfolding and extension by telomerase. 13These data reveal that telomerase is a parallel G-quadruplex resolvase.14 Human chromosomes contain many guanine (G)-rich elements capable of forming four-1 stranded G-quadruplex (G4) structures (1-4). A planar G-quartet is formed when four Gs form 2 hydrogen bonds in a cyclical manner via their Hoogsteen faces; such G-quartets stack on top 3 of each other and form G-quadruplexes (5, 6). G-rich sequences are primarily located in 4 promoter regions, intron and exon boundaries, origins of replication and telomeres (1-4). 5Vertebrate telomeres consist of many tandem repeats of the sequence TTAGGG, comprising a 6 double-stranded region and a 3' G-rich overhang (7). Fluorescence microscopy studies have 7 reached, telomerase translocates downstream, resulting in re-alignment of the RNA template 1 with the new 3' end of the product DNA (19, 22). 2One function of telomeric G4 may be to act as a 'cap', protecting the telomere from DNA 3 degradation (24). It has been widely believed that G4 formation within the 3' telomeric 4 overhang blocks telomere extension by telomerase, since early in vitro studies had shown that 5 G4 structures inhibit telomerase extension of a telomeric DNA substrate (25,26). In addition, 6 stabilization of G4 with small-molecule ligands has been shown to more effectively inhibit 7 telomerase activity, suggesting that chemical stabilization of G4 structures may be a viable 8 anti-cancer therapeutic strategy (27)(28)(29). However, the above-mentioned studies did not 9 distinguish between G4 conformations, and used oligonucleotides that likely folded into anti-10 parallel or 'hybrid' G4 forms as a telomerase substrate. A variety of helicases, such as RECQ5, 11 WRN, BLM, FANC-J and RHAU, recognize and resolve G4 in a conformation-specific 12 manner (30)(31)(32)(33). Similarly, we have demonstrated that telomerase can bind and extend specific 13 conformations of telomeric G4 -those t...

G-Quadruplexes at Telomeres: Friend or Foe?

“…However, not all G-quadruplexes are refractory to telomerase extension. Parallel G-quadruplexes, whether intermolecular or intramolecular, can be bound and extended by both ciliate and human telomerase [ 39 , 155 , 159 , 160 ]. The 3′ end of the DNA aligns correctly with the RNA template of telomerase [ 39 , 159 ], and telomerase then unwinds the rest of the G-quadruplex as it extends the DNA [ 160 ].…”

“…Parallel G-quadruplexes, whether intermolecular or intramolecular, can be bound and extended by both ciliate and human telomerase [ 39 , 155 , 159 , 160 ]. The 3′ end of the DNA aligns correctly with the RNA template of telomerase [ 39 , 159 ], and telomerase then unwinds the rest of the G-quadruplex as it extends the DNA [ 160 ]. It should be noted, however, that the preferred substrate of telomerase is linear DNA; the affinity for parallel G-quadruplexes is about 5-fold lower than their corresponding linear oligonucleotides, and accommodation of the relatively bulky G-quadruplex in the active site results in a decrease in the affinity of telomerase for incoming nucleotides [ 39 , 159 ].…”

“…This may not necessarily be a bad thing, since telomere uncapping or genomic DNA damage may cause more rapid effects on cancer cell viability than the delayed senescence caused by gradual telomere shortening. Thirdly, human telomerase does have the ability to extend some G-quadruplexes [ 39 ], and its ability to do so is not always inhibited by G4-stabilizing ligands [ 160 ]. Therefore, recent attention has focused on nontelomeric mechanisms by which G4-stabilizing molecules may cause death or growth arrest of cancer cells [ 168 , 169 , 170 ].…”

An Updated Focus on Quadruplex Structures as Potential Therapeutic Targets in Cancer

“…However, hTERT is able to extend parallel, intermolecular G4s conformations in vitro and this ability is conserved among evolutionarily distant species [ 92 ]. In fact, recent data reveal that hTERT acts as a parallel G4 resolvase [ 93 ]. In addition, hTERT is involved in mitochondrial apoptosis induced by targeted inhibition of BCL2 [ 94 ], regulates the chromatin state and DNA damage response [ 95 ] and promotes c-MYC and WNT-driven cellular proliferation [ 96 ].…”

Characterization of In Vitro G-Quadruplex Formation of Imetelstat Telomerase Inhibitor

“…Several synthetic G-quadruplex stabilizing small molecules such as telomestatin, BRACO-19, and TMPyP4, have demonstrated potential anticancer properties [21,22]. Recently however, several reports established that human telomerase could recognize, unwind, and extend intra-and interparallel G-quadruplex conformations in vitro and that they are colocalizing in vivo [14,23]. Moreover, Jansson et al recently demonstrated that G-quadruplexes can form within actively extending telomerase [24].…”