‘If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo’ (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.

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Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3 -terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3 -overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd ‫؍‬ 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd ‫؍‬ 3-5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.T elomeres are specialized DNA-protein complexes at the end of eukaryotic chromosomes. They protect the chromosome ends from recombination, from fusion, and from being mistaken as broken ends (1-3). Telomeric DNA consists of simple repetitive DNA sequences ranging in size from 36 nucleotides in hypotrichous ciliates up to 50 kb in mammals. The 3Ј-strand is usually G-rich and extends over the complementary strand. It has been shown that the G-rich overhang can adopt a variety of unusual DNA structures (4), of which guanine-quadruplex DNA and t-loops are stable in vitro under physiological conditions (5-8). Parallel-stranded as well as antiparallel-stranded guaninequadruplex structures have been biophysically and structurally analyzed in detail with synthetic oligonucleotides (6-11). It has been suggested that this structure is also involved in numerous cellular processes, including transcription and recombination, in addition to telomere function (12). However, direct evidence for this DNA structure in vivo has been lacking to date.Ciliated protozoa are a well-suited biological system to study telomere structure and function, and many important processes including telomere sequences and telomerase were first identified in these cells (1-3). Hypotrichous ciliates, such as Oxytricha, Euplotes, or Styl...

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPalpha and TEBPbeta. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPalpha in attaching telomeres in the nucleus and in recruiting TEBPbeta to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPbeta in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPbeta.