ReviewIn vivo veritas: Using yeast to probe the biological functions of G-quadruplexes
Introduction
The genomes and transcriptomes of many organisms, including those as diverse as E. coli and humans, contain a number of G-rich sequences that, at least in vitro and perhaps in vivo, are capable of forming structures known as G-quadruplexes (G4-DNA and G4-RNA, respectively). These structures are composed of stacked associations of G-quartets, which are planar assemblies of four Hoogsteen-bonded guanines (Fig. 1A and B) [1], [2]. G4 structures can arise through the interactions of guanines present on a single nucleic acid strand (intra-molecular) or multiple strands (inter-molecular). Beyond hydrogen bonding among guanines, the stability of quadruplexes derives from π-orbital interactions among stacked quartets as well as coordination by quartets of centrally located cations (e.g. Na+ or K+). Thus a minimum of two adjacent quartets, but ideally three or more, is required for stable quadruplex formation. G4 structures are stable under physiologic salt and pH conditions in vitro, and some have higher melting temperatures than the duplex DNA that would be formed by providing the complementary strand. There is a high degree of polymorphism among different G4 structures. In principal, 16 different quartet structures can form, which are distinguished by the patterns of glycosidic bond angles of the guanines [3]. Further, the number of stacked quartets, the number and polarity of the phosphodiester backbone strands from which the guanines extend, the type of coordinated cations, and the length, sequence and connectivity of intervening loops may vary [1].
Although the structures of G-quadruplexes have been well studied in vitro, if, when and where they form in vivo and how they might affect cell biology have remained key questions. The structural heterogeneity of quadruplexes makes it difficult to obtain universal rules to predict their formation or probes to test for their presence. Nonetheless, a good deal of information demonstrating or strongly suggesting their functions in vivo has emerged in recent years. For example, telomeric G4-DNA has been proven to exist in Stylonichia lemnae [4], [5], and sequences with intramolecular quadruplex-forming potential (QFP) have been shown to be highly overrepresented in the promoter regions of diverse organisms and to be connected with control of gene expression [6], [7], [8], [9], [10], [11], [12]. In addition, a number of small molecule ligands have been identified that bind to and stabilize quadruplexes (Fig. 1C) [13], [14], [15], [16], [17], [18], [19], [20], [21], and some of these have been found to affect expression from QFP-containing loci, indicating that QFP sequences can adopt G4 conformations.
Here we review findings pertaining to the in vivo functions of G-quadruplexes, with an emphasis on findings in yeast. We begin by highlighting the ways in which yeast model systems can help identify and dissect the cellular roles of quadruplex structures. For readers particularly interested in findings outside of yeast, we recommend these outstanding reviews [22], [23]. Yeast genetic tools have significant potential for revealing the full extent to which G-quadruplexes regulate biological processes, as well as for revealing underlying mechanisms.
Section snippets
Genetic systems for the analysis of G-quadruplexes in yeast
S. cerevisiae offers several genetic systems that could facilitate exploration of the in vivo functions of G-quadruplexes. Although none is unique to yeast, the ease with which they can be carried out in this single celled eukaryote make it an ideal choice for these studies. We first describe these systems, and in the second half of the review describe findings obtained from their use.
Evidence for G-quadruplex functions in vivo
Many observations suggest, and in some cases demonstrate, roles for G-quadruplexes in different aspects of cell biology. Each of the sections below describes one such aspect, beginning with general examples from several organisms and then focusing on findings from yeast. Table 1 provides a summary of various yeast proteins implicated in G-quadruplex metabolism.
Perspective
A combination of biophysical, bioinformatic, genetic and cell biological approaches have yielded a remarkable series of findings that argue for the relevance of G-quadruplexes to natural biology. However, more work is required to firmly establish the roles of G4-DNA and G4-RNA in nucleic acid functions and to decipher the mechanisms by which they operate. This knowledge might provide new approaches for selectively targeting processes ranging from transcription and translation, to DNA
Acknowledgements
We thank Li-San Wang, Steve Hershman and Qijun Chen for discussions, and Alex Chavez for insightful comments on the manuscript. This work was supported by the National Institute on Aging (5R01AG021521 to F.B.J).
References (120)
- et al.
Enrichment of G4 DNA motif in transcriptional regulatory region of chicken genome
Biochem. Biophys. Res. Commun.
(2007) - et al.
Targeting telomeres and telomerase
Biochimie.
(2008) - et al.
G-quadruplexes as targets for drug design
Pharmacol. Ther.
(2000) - et al.
dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae
Methods
(2007) - et al.
Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile
Cell
(2005) - et al.
Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast
Cell
(2006) - et al.
A conserved G4 DNA binding domain in RecQ family helicases
J. Mol. Biol.
(2006) - et al.
Use of competition dialysis in the discovery of G-quadruplex selective ligands
Methods
(2007) - et al.
The beta subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA
Cell
(1993) - et al.
Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein
Cell
(2004)