In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.

G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by non-canonical G-G base pairs. Genome-wide chromatin immuno-precipitation was used to determine the in vivo binding sites of the multi-functional S. cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high confidence Pif1 binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, while non G4 Pif1 binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication stressed Pif1 deficient cells that was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication and stimulate DNA breakage. These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage.

The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. We show that exceptionally potent G-quadruplex unwinding is conserved amongst Pif1 helicases. Moreover, Pif1 helicases from organisms separated by >3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to novel genetic and epigenetic changes. Further, when expressed in yeast, human Pif1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.

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.

G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

DNA and RNA can fold into a variety of alternative conformations. In recent years, a particular nucleic acid structure was discussed to play a role in malignant transformation and cancer development. This structure is called a G-quadruplex (G4). G4 structure formation can drive genome instability by creating mutations, deletions and stimulating recombination events. The importance of G4 structures in the characterization of malignant cells was currently demonstrated in breast cancer samples. In this analysis a correlation between G4 structure formation and an increased intratumor heterogeneity was identified. This suggests that G4 structures might allow breast cancer stratification and supports the identification of new personalized treatment options. Because of the stability of G4 structures and their presence within most human oncogenic promoters and at telomeres, G4 structures are currently tested as a therapeutic target to downregulate transcription or to block telomere elongation in cancer cells. To date, different chemical molecules (G4 ligands) have been developed that aim to target G4 structures. In this review we discuss and compare G4 function and relevance for therapeutic approaches and their impact on cancer development for three cancer entities, which differ significantly in their amount and type of mutations: pancreatic cancer, leukemia and malignant melanoma. G4 structures might present a promising new strategy to individually target tumor cells and could support personalized treatment approaches in the future.

Summary Human RecQ4 affects cancer and aging, but it is difficult to study because it is a fusion between a helicase and an essential replication factor. Budding yeast Hrq1 is homologous to the disease-linked helicase domain of RecQ4 and, like hRecQ4, was a robust 3’–5’ helicase. Additionally, Hrq1 had the unusual property of forming heptameric rings. Cells lacking Hrq1 exhibited two particularly dangerous DNA damage phenotypes: hypersensitivity to DNA inter-strand crosslinks (ICLs) and telomere addition to DNA breaks. Both activities are rare: their co-existing in a single protein is unprecedented. Resistance to ICLs required helicase activity, but suppression of telomere addition did not. Hrq1 also affected telomere length by a non-catalytic mechanism, as well as telomerase-independent telomere maintenance. As Hrq1 bound telomeres in vivo, it likely affects them directly. Thus, the tumor suppressing activity of RecQ4 could be due to a role in ICL repair and/or suppressing de novo telomere addition.

The concept that G-quadruplex (G4) structures can form within DNA or RNA has been long known and extensively discussed. In recent years, accumulating evidences imply that G-quadruplex structures form Initially, inefficient regulation of G-quadruplex structures was mainly associated with genome instability. However, due to the location of G-quadruplex motifs and their evolutionary conservation, different cellular functions of these structures have been postulated (e.g. in telomere maintenance, DNA replication, transcription, and translation). Regardless of their function, efficient and controlled formation and unwinding are very important, because 'mis'-regulated G-quadruplex structures are detrimental for a given process, causing genome instability and diseases. Several helicases have been shown to target and regulate specific G-quadruplex structures. This mini-review focuses on the biological consequences of G4 disruption by different helicases .