Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Fast, sensitive, and specific multiplexed single-molecule detection of circulating tumor DNA.

Circulating tumor DNA (ctDNA) analysis has emerged as a highly promising non-invasive assay for detection and monitoring of cancer. However, identification of multiple point-mutant ctDNAs, particularly at extremely low frequencies in early cancer stages, remains a significant challenge. To address this issue, we present a multiplexed ctDNA detection technique, SIMUL (single-molecule detection of multiple low-frequency mutations). SIMUL involves an unbiased preamplification of both wild-type and mutant DNAs, followed by the detection of mutant DNAs through single-molecule multicolor imaging. SIMUL enables highly sensitive and specific detection of multiple single-nucleotide mutations in a short span of time, even in the presence of 10,000-fold excess of wild-type DNA. Importantly, SIMUL can accurately measure mutant fractions due to its linear correlation between the number of single-molecule spots and the variant allele frequency. This breakthrough technique holds immense potential for clinical applications, offering significant improvements for example in early cancer detection and accurate evaluation of anticancer treatment responses.

Rapid quantification of miRNAs using dynamic FRET-FISH.

MicroRNAs (miRNAs) are short regulatory RNAs that control gene expression at the post-transcriptional level. Various miRNAs playing important roles in cancer development are emerging as promising diagnostic biomarkers for early cancer detection. Accurate miRNA detection, however, remains challenging because they are small and highly homologous. Recently developed miRNA detection techniques based on single-molecule imaging enabled highly specific miRNA quantification without amplification, but the time required for these techniques to detect a single miRNA was larger than 10 minutes, making rapid profiling of numerous miRNAs impractical. Here we report a rapid miRNA detection technique, dynamic FRET-FISH, in which single-molecule imaging at high probe concentrations and thus high-speed miRNA detection is possible. Dynamic FRET-FISH can detect miRNAs in 10 s at 1.2 µM probe concentration while maintaining the high-specificity of single-nucleotide discrimination. We expect dynamic FRET-FISH will be utilized for early detection of cancers by profiling hundreds of cancer biomarkers in an hour.

A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity.

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

Yeast Chd1p Unwraps the Exit Side DNA upon ATP Binding to Facilitate the Nucleosome Translocation Occurring upon ATP Hydrolysis.

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. We use single-molecule fluorescence experiments to clarify the mechanism by which yeast CHD1 (Chd1p) remodels nucleosomes. We find that binding of ATP to Chd1p induces transient unwrapping of the DNA on the exit side of the nucleosome, facilitating nucleosome translocation. ATP hydrolysis is required to induce nucleosome translocation. The unwrapped DNA after translocation is then rewrapped after the release of the hydrolyzed nucleotide and phosphate, revealing that each step of the ATP hydrolysis cycle is responsible for a distinct step of nucleosome remodeling. These results show that Chd1p remodels nucleosomes via a mechanism that is unique among the other ATP-dependent chromatin remodelers.

Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation.

G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

ATP Binding to Rad5 Initiates Replication Fork Reversal by Inducing the Unwinding of the Leading Arm and the Formation of the Holliday Junction.

Replication fork reversal is one of the major pathways for reactivating stalled DNA replication. Many enzymes with replication fork reversal activity have DNA-unwinding activity as well, but none of the fork reversal enzymes in the SWI/SNF family shows a separate DNA-unwinding activity, raising the question of how they initiate the remodeling process. Here, we found ATP binding to Rad5 induces the unwinding of the leading arm of the replication fork and proximally positions the leading and lagging arms. This facilitates the spontaneous remodeling of the replication fork into a four-way junction. Once the four-way junction is formed, Rad5 migrates the four-way junction at a speed of 7.1 ± 0.14 nt/s. The 3' end anchoring of the leading arm by Rad5's HIRAN domain is critical for both branch migration and the recovery of the three-way junction, but not for the structural transition to the four-way junction.

Extended depth of field for single biomolecule optical imaging-force spectroscopy.

Real-time optical imaging combined with single-molecule manipulation broadens the horizons for acquiring information about the spatiotemporal localization and the mechanical details of target molecules. To obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy, we developed an optical method to extend the depth of field in a high numerical aperture objective (≥ 1.2), required to visualize a single fluorophore. By axial scanning, using an electrically tunable lens with a fixed sample, we were successfully able to visualize the epidermal growth factor receptor (EGFR) moving along the three-dimensionally elongated filamentous actin bundles connecting cells (intercellular nanotube), while another EGFR on the intercellular nanotube was trapped by optical tweezers in living cells. Our approach is simple, fast and inexpensive, but it is powerful for imaging target molecules axially in single-molecule optical imaging-force spectroscopy.

NAP1L1 accelerates activation and decreases pausing to enhance nucleosome remodeling by CSB.

Cockayne syndrome protein B (CSB) belongs to the SWI2/SNF2 ATP-dependent chromatin remodeler family, and CSB is the only ATP-dependent chromatin remodeler essential for transcription-coupled nucleotide excision DNA repair. CSB alone remodels nucleosomes ∼10-fold slower than the ACF remodeling complex. Strikingly, NAP1-like histone chaperones interact with CSB and greatly enhance CSB-mediated chromatin remodeling. While chromatin remodeling by CSB and NAP1-like proteins is crucial for efficient transcription-coupled DNA repair, the mechanism by which NAP1-like proteins enhance chromatin remodeling by CSB remains unknown. Here we studied CSB's DNA-binding and nucleosome-remodeling activities at the single molecule level in real time. We also determined how the NAP1L1 chaperone modulates these activities. We found that CSB interacts with DNA in two principle ways: by simple binding and a more complex association that involves gross DNA distortion. Remarkably, NAP1L1 suppresses both these interactions. Additionally, we demonstrate that nucleosome remodeling by CSB consists of three distinct phases: activation, translocation and pausing, similar to ACF. Importantly, we found that NAP1L1 promotes CSB-mediated remodeling by accelerating both activation and translocation. Additionally, NAP1L1 increases CSB processivity by decreasing the pausing probability during translocation. Our study, therefore, uncovers the different steps of CSB-mediated chromatin remodeling that can be regulated by NAP1L1.

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase.

The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3.2 ± 0.03 bases/s velocity. The recovery of the replication fork was controlled by branch migration reversal of WRN, resulting in repetitive fork regression. The Holliday junction formation, branch migration, and migration direction reversal are all ATP dependent, revealing that WRN uses the energy of ATP hydrolysis to actively coordinate the structural transitions of DNA.

Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments.

The viral sensor MDA5 distinguishes between cellular and viral dsRNAs by length-dependent recognition in the range of ~0.5-7 kb. The ability to discriminate dsRNA length at this scale sets MDA5 apart from other dsRNA receptors of the immune system. We have shown previously that MDA5 forms filaments along dsRNA that disassemble upon ATP hydrolysis. Here, we demonstrate that filament formation alone is insufficient to explain its length specificity, because the intrinsic affinity of MDA5 for dsRNA depends only moderately on dsRNA length. Instead, MDA5 uses a combination of end disassembly and slow nucleation kinetics to "discard" short dsRNA rapidly and to suppress rebinding. In contrast, filaments on long dsRNA cycle between partial end disassembly and elongation, bypassing nucleation steps. MDA5 further uses this repetitive cycle of assembly and disassembly processes to repair filament discontinuities, which often are present because of multiple, internal nucleation events, and to generate longer, continuous filaments that more accurately reflect the length of the underlying dsRNA scaffold. Because the length of the continuous filament determines the stability of the MDA5-dsRNA interaction, the mechanism proposed here provides an explanation for how MDA5 uses filament assembly and disassembly dynamics to discriminate between self vs. nonself dsRNA.

DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg2+-mediated dynamic bending of gate-DNA.

Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg(2+) ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N-gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.