G-quadruplex (G4) can be formed by G-rich DNA sequences that are widely distributed throughout the human genome. Although G-triplex and G-hairpin have been proposed as G4 folding intermediates, their formation still requires further investigation by experiments. Here, we employed single-molecule FRET to characterize the folding dynamics of G4 from human telomeric sequence. First, we observed four states during G4 folding initially assigned to be anti-parallel G4, G-triplex, G-hairpin and unfolded ssDNA. Then we constructed putative intra-strand G-triplex, G-hairpin structures and confirmed their existences in both NaCl and KCl. Further studies revealed those structures are going through dynamic transitions between different states and show relatively weak dependence on cations, unlike G4. Based on those results and molecular dynamics simulations, we proposed a multi-pathway folding mechanism for human telomeric G4. The present work may shed new light on our current understanding about the existence and stability of G4 intermediate states.

Mutations in the RecQ DNA helicase gene BLM give rise to Bloom's syndrome, which is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition. BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients. With smFRET assays, we studied the molecular mechanism of BLM-catalyzed G4 unfolding and showed that ATP is required for G4 unfolding. Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3'-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA. This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.

Recent advances in G-quadruplex (G4) studies have confirmed that G4 structures exist in living cells and may have detrimental effects on various DNA transactions. How helicases resolve G4, however, has just begun to be studied and remains largely unknown. In the present paper, we use single-molecule fluorescence assays to probe Pif1-catalysed unfolding of G4 in a DNA construct resembling an ongoing synthesis of lagging strand stalled by G4. Strikingly, Pif1 unfolds and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and ‘acrobatic’ re-initiation of unfolding by the same monomer. Thus, Pif1 unfolds single G4 structures repetitively. Furthermore, it is found that Pif1 unfolds G4 sequentially in two large steps. Our study has revealed that, as a stable intermediate, G-triplex (G3) plays an essential role in this process. The repetitive unfolding activity may facilitate Pif1 disrupting the continuously reforming obstructive G4 structures to rescue a stalled replication fork. The proposed mechanism for step-wise unfolding of G4 is probably applicable to other helicases that resolve G4 structures for maintaining genome stability.

The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p^237−780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.

The unique dumbbell-shape of grass guard cells (GCs) is controlled by their cell walls which enable their rapid responses to the environment. The molecular mechanisms regulating the synthesis and assembly of GC walls are as yet unknown. Here we have identified BZU3, a maize gene encoding UDP-glucose 4-epimerase that regulates the supply of UDP-glucose during GC wall synthesis. The BZU3 mutation leads to significant decreases in cellular UDP-glucose levels. Immunofluorescence intensities reporting levels of cellulose and mixed-linkage glucans are reduced in the GCs, resulting in impaired local wall thickening. BZU3 also catalyzes the epimerization of UDP-N-acetylgalactosamine to UDP-N-acetylglucosamine, and the BZU3 mutation affects N-glycosylation of proteins that may be involved in cell wall synthesis and signaling. Our results suggest that the spatiotemporal modulation of BZU3 plays a dual role in controlling cell wall synthesis and glycosylation via controlling UDP-glucose/N-acetylglucosamine homeostasis during stomatal morphogenesis. These findings provide insights into the mechanisms controlling formation of the unique morphology of grass stomata.