G4 DNA: at risk in the genome.

Regions bearing G-quadruplex (G4) DNA motifs can be sites of genomic instability and are frequently depleted in streamlined genomes, but are nevertheless maintained in many other genomes. Whether G4 secondary structures form in vivo, and how they are maintained or eliminated, remains little known. In this issue of The EMBO Journal, Lopes et al (2011) provide new evidence that G4 structures form and contribute to genomic instability in living cells, and identify specific mechanisms that minimize the risks posed by G4 motifs. G4 DNA structures can form within regions bearing at least four runs of guanines, with at least three guanines per run (GX3NxGX3NxGX3NxGX3). In the human genome, the rDNA, telomeres, and immunoglobulin switch regions are rich in G4 sequence motifs, as are some highly unstable minisatellites and single copy genes. Evidence that G4 structures can form within these regions has been provided by single molecule imaging, immunofluorescence microscopy, and genetic and biochemical characterization of factors that bind and unwind G4 DNA (reviewed by Maizels, 2006). Conservation of both position and structural potential, though not necessarily of sequence, suggests that G4 structures participate in key cellular processes. G4 structures at specific sites are intracellular targets of regulatory processes, including control of pilin antigen variation in Neisseria gonorrhoeae (Cahoon and Seifert, 2009), and p53 mRNA 30-end formation in stressed cells (Decorsiere et al, 2011). G4 structures have considerable thermal stability and could block progression of both DNA and RNA polymerases if not resolved. Cellular helicases that actively unwind G4 DNA may facilitate polymerase progression. RecQ family helicases (human BLM and WRN, Escherichia coli RecQ) share a conserved RQC domain that promotes G4 recognition, and unwind with 30–50 directionality. Werner syndrome, a human genetic disease characterized by premature ageing, is caused by WRN helicase deficiency, and cells lacking WRN exhibit sequence loss at telomeres at which the G-rich strand is the template for lagging strand replication (Crabbe et al, 2004). FANCJ-related helicases (human FANCJ, nematode dog-1) are DEAH/X superfamily II helicases, and unwind with 50–30 directionality. In nematodes deficient in dog-1, long DNA deletions with the 30-end defined by G4 motifs occur (Kruisselbrink et al, 2008), and patient cells deficient in FANCJ echo this phenotype. The new paper by Nicolas and collaborators (Lopes et al, 2011) combines genetic, chemical, and physical approaches to show that, in Saccharomyces cerevisiae, G4 DNA formed on the template for leading strand replication is normally unwound by Pif1, a 50–30 helicase related to E. coli RecD. These experiments take advantage of an elegant S. cerevisiae-based reporter assay, developed previously by the Nicolas laboratory, that quantifies the frequency of size variants of the human CEB1 minisatellite. The CEB1 repeat, which is rich in G4 motifs and highly polymorphic in human populations,