G-quadruplexes and their regulatory roles in biology

Abstract

'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.

Figure 1.

Structure of G-quadruplexes. G-quadruplexes form in vitro in DNA or RNA sequences containing tracts of three to four guanine. (A) The building blocks of G-quadruplexes are G-quartets that arise from the association of four guanines into a cyclic arrangement stabilized by Hoogsten hydrogen bonding (N1–N6 and N2–N7). The planar G-quartets stack on top of one another, forming four-stranded helical structures. G-quadruplex formation is driven by monovalent cations such as Na⁺ and K⁺. (B) G-quadruplex structures are polymorphic and can be sub-grouped into different families, as for example parallel or antiparallel according to the orientation of the strands and can be inter- or intramolecular folded. The type of structure depends on the number of G-tracts in a strand.

Figure 2.

Possible locations of G-quadruplex structures in cells. Genome wide searches have revealed the location of G-rich regions with G-quadruplex forming potential (pG4). pG4s are non-randomly distributed in the genome and promoters and telomeres are particularly enriched in these sequences. In the nucleus, G-quadruplex formation can occur in double stranded G-rich regions when DNA becomes transiently single stranded, during (A) transcription and (C) replication and (B) at the single stranded telomeric G-rich overhangs. Outside the nucleus, G-quadruplexes can also form in mRNA and (D) are involved in translational control. Red T-bars indicate impediments to transcription, replication and translation.

Figure 3.

G-quadruplexes at telomeres. The G-rich overhang of telomeres can form G-quadruplex structures involved in telomere end protection and telomeric DNA metabolism. (A) The long human G-rich overhang can form strings intramolecularly folded G-quadruplexes that may offer end protection against nucleases or regulate telomerase activity. (B) Ciliate telomeres form intermolecular G-quadruplex structures involving two telomeres promoted by the telomere-end binding protein TEBPβ. Telomeres are attached to a sub-nuclear structure (the nuclear matrix or scaffold) via an interaction of the telomere-end binding protein TEBPα. (C) Stabilizing of G-quadruplexes by G-quadruplex binding ligands (yellow stars) impairs telomere repeat synthesis by the telomerase enzyme and lead to telomere shortening (modified after (80)).

Figure 4.

G-quadruplexes in transcription and translation. (A) pG4 sequences are present in about 50% of human genes promoters. G-quadruplex formation could impair initiation of transcription by the RNA polymerase, or if present in the antisense strand inhibit transcription. (B) The presence of G-quadruplexes formed in the 5′ UTR of mRNAs can regulate translation as well as lead to aborted RNA transcripts in hexanucleotide repeat expansion diseases.

Figure 5.

G-quadruplexes and replication. G-quadruplexes formed during replication when the DNA is transiently single stranded impede replication and have to be resolved to permit the replication machinery including DNA polymerase to proceed for both leading and lagging strand DNA synthesis. G-quadruplexes are known to be resolved by G-quadruplex unwinding helicases such as FANCJ that has a 5′–3′ directionality. Other helicases such as BLM and WRN have a 3′–5′ directionality. Other proteins or enzymes such as polymeras also function in the successful bypass of G-quadruplexes.

Figure 6.

G-quadruplexes and the initiation of DNA replication. Origins of replication in mice and humans are GC-rich and contain pG4s. G-quadruplex formation is required for the initiation of DNA replication and the localization of the G-quadruplex determines the site of initiation (modified after (117)).