Ja Yil Lee

DNA with tandem repeats of guanines folds into G-quadruplexes made of a stack of G-quartets. In vitro, G-quadruplex formation inhibits telomere extension, and POT1 binding to the single-stranded telomeric DNA enhances telomerase activity by disrupting the G-quadruplex structure, highlighting the potential importance of the G-quadruplex structure in regulating telomere length in vivo. We have used single-molecule spectroscopy to probe the dynamics of human telomeric DNA. Three conformations were observed in potassium solution, one unfolded and two folded, and each conformation could be further divided into two species, long-lived and short-lived, based on lifetimes of minutes vs. seconds. Vesicle encapsulation studies suggest that the total of six states detected here is intrinsic to the DNA. Folding was severely hindered by replacing a single guanine, showing only the shortlived species. The long-lived folded states are dominant in physiologically relevant conditions and probably correspond to the parallel and antiparallel G-quadruplexes seen in high-resolution structural studies. Although rare under these conditions, the short-lived species determine the overall dynamics because they bridge the different long-lived species. We propose that these previously unobserved transient states represent the early and late intermediates toward the formation of stable G-quadruplexes. The major compaction occurs between the early and late intermediates, and it is possible that local rearrangements are sufficient in locking the late intermediates into the stably folded forms. The extremely diverse conformations of the human telomeric DNA may have mechanistic implications for the proteins and drugs that recognize G-rich sequences.

DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination.

Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p. Small forces on the filaments dramatically slowed formin-mediated polymerization in the absence of profilin, but resulted in faster polymerization in the presence of profilin. We propose that force shifts the conformational equilibrium of the end of a filament associated with formin homology 2 domains toward the closed state that precludes polymerization, but that profilin-actin associated with formin homology 1 domains reverses this effect. Thus, physical forces strongly influence actin assembly by formin Bni1p.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.