The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer.

Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes with sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription.

Probing Dynamic Assembly and Disassembly of Rad51 Tuned by Srs2 Using smFRET.

The integrity of DNA is critical for sustaining the life of any living organism, as DNA is a reservoir of its genetic information. However, DNA is continuously damaged by either normal metabolic pathways or environmental insults such as ultraviolet exposure or chemicals. Double-stranded DNA break is one of the most common types of DNA damage that requires activation of homologous recombination (HR) pathway mediated by Rad51 in eukaryotes (Paques & Haber, 1999; Symington, 2002). Rad51 protein forms a helical nucleoprotein filament on resected DNA to initiate homology search but also can interact with other single-stranded DNA (ssDNA)-binding proteins including Srs2. Srs2, a well-known antirecombinase in HR, is an ATP-dependent 3'-5' DNA helicase in the budding yeast Saccharomyces cerevisiae as well as an ssDNA translocase. It disrupts Rad51 filaments, preventing HR (Krejci et al., 2003; Le Breton et al., 2008; Veaute et al., 2003). In the following text, we provide detailed experimental platforms employed to investigate the activity of Rad51 and Srs2 using single-molecule Forster resonance energy transfer and protein-induced fluorescence enhancement. First, we demonstrate how to detect Rad51 filament formation to address the binding site size binding kinetic of the Rad51, as well as the directionality of the filament formation. Next, we explain how to visualize ATP-dependent translocation and unwinding activities of Srs2 on DNA. Lastly, we demonstrate the filament forming activity by Rad51 which is counteracted by the filament removal activity of Srs2.

A guanine-flipping and sequestration mechanism for G-quadruplex unwinding by RecQ helicases.

Homeostatic regulation of G-quadruplexes (G4s), four-stranded structures that can form in guanine-rich nucleic acids, requires G4 unwinding helicases. The mechanisms that mediate G4 unwinding remain unknown. We report the structure of a bacterial RecQ DNA helicase bound to resolved G4 DNA. Unexpectedly, a guanine base from the unwound G4 is sequestered within a guanine-specific binding pocket. Disruption of the pocket in RecQ blocks G4 unwinding, but not G4 binding or duplex DNA unwinding, indicating its essential role in structure-specific G4 resolution. A novel guanine-flipping and sequestration model that may be applicable to other G4-resolving helicases emerges from these studies.

mRNA structure determines specificity of a polyQ-driven phase separation.

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

Molecular mechanism of resolving trinucleotide repeat hairpin by helicases.

Trinucleotide repeat (TNR) expansion is the root cause for many known congenital neurological and muscular disorders in human including Huntington's disease, fragile X syndrome, and Friedreich's ataxia. The stable secondary hairpin structures formed by TNR may trigger fork stalling during replication, causing DNA polymerase slippage and TNR expansion. Srs2 and Sgs1 are two helicases in yeast that resolve TNR hairpins during DNA replication and prevent genome expansion. Using single-molecule fluorescence, we investigated the unwinding mechanism by which Srs2 and Sgs1 resolves TNR hairpin and compared it with unwinding of duplex DNA. While Sgs1 unwinds both structures indiscriminately, Srs2 displays repetitive unfolding of TNR hairpin without fully unwinding it. Such activity of Srs2 shows dependence on the folding strength and the total length of TNR hairpin. Our results reveal a disparate molecular mechanism of Srs2 and Sgs1 that may contribute differently to efficient resolving of the TNR hairpin.

Context-dependent remodeling of Rad51-DNA complexes by Srs2 is mediated by a specific protein-protein interaction.

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3' ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.

Srs2 prevents Rad51 filament formation by repetitive motion on DNA.

Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation.

Ultrasonic viscoelasticity imaging of nonpalpable breast lesions.

The prognosis of breast cancer patients improves with early and accurate diagnosis. A small clinical study was conducted with 21 women having a single nonpalpable breast lesion, each detected mammographically with later pathology confirmation. Elasticity images were acquired on each patient to test for the ability to differentiating malignant and benign lesions. The mechanical relaxation time T(1) images showed a tissue-specific T(1) contrast that is negative for all 11 malignant lesions and positive for all 10 benign lesions. Strain images were estimated using a regularized multi-scale optical flow (ROF) algorithm. Adjustments to the input parameters to the ROF and their subsequent effects on T(1) estimation and computation time are shown to have a strong effect of diagnostic performance.

Ultrasonic viscoelasticity imaging of nonpalpable breast tumors: preliminary results.

RATIONALE AND OBJECTIVES: Improvements in the diagnosis of early breast cancers depend on a physician's ability to obtain the information necessary to distinguish nonpalpable malignant and benign tumors. Viscoelastic features that describe mechanical properties of tissues may help to distinguish these types of lesions. MATERIALS AND METHODS: Twenty-one patients with nonpalpable, pathology-confirmed Breast Imaging Reporting and Data System (BIRADS) 4 or 5 breast lesions (10 benign, 11 malignant) detected by mammography were studied. Viscoelastic parameters were extracted from a time sequence of ultrasonic strain images, and differences in the parameters between malignant and benign tumors were compared. Parametric data were color coded and superimposed on sonograms. RESULTS: The strain retardance time parameter, T(1), provided the best discrimination between malignant and benign tumors (P < .01). T(1) measures the time required for tissues to fully deform (strain) once compressed; therefore, it describes the time-varying viscous response of tissue to a small deforming force. Compared to the surrounding background tissues, malignant lesions have smaller average T(1) values, whereas benign lesions have higher T(1) values. This tissue-specific contrast correlates with known changes in the extracellular matrix of breast stroma. CONCLUSION: Characterization of nonpalpable breast lesions is improved by the addition of viscoelastic strain imaging parameters. The differentiation of malignant and benign BI-RADS 4 or 5 tumors is especially evident with the use of the retardation time estimates, T(1).

Integrated endoscope for real-time 3D ultrasound imaging and hyperthermia: feasibility study.

The goal of this research is to determine the feasibility of using a single endoscopic probe for the combined purpose of real-time 3D (RT3D) ultrasound imaging of a target organ and the delivery of ultrasound therapy to facilitate the absorption of compounds for cancer treatment. Recent research in ultrasound therapy has shown that ultrasound-mediated drug delivery improves absorption of treatments for prostate, cervical and esophageal cancer. The ability to combine ultrasound hyperthermia and 3D imaging could improve visualization and targeting of cancerous tissues. In this study, numerical modeling and experimental measurements were developed to determine the feasibility of combined therapy and imaging with a 1 cm diameter endoscopic RT3D probe with 504 transmitters and 252 receive channels. This device operates at 5 MHz and has a 6.3 mm x 6.3 mm aperture to produce real time 3D pyramidal scans of 60-120 degrees incorporating 64 x 64 = 4096 image lines at 30 volumes/sec interleaved with a 3D steerable therapy beam. A finite-element mesh was constructed with over 128,000 elements in LS-DYNA to simulate the induced temperature rise from our transducer with a 3 cm deep focus in tissue. Quarter-symmetry of the transducer was used to reduce mesh size and computation time. Based on intensity values calculated in Field II using the transducer's array geometry, a minimum I(SPTA) of 3.6 W/cm2 is required from our endoscope probe in order to induce a temperature rise of 4 degrees C within five minutes. Experimental measurements of the array's power output capabilities were conducted using a PVDF hydrophone placed 3 cm away from the face of the transducer in a watertank. Using a PDA14 Signatec data acquisition board to capture full volumes of transmitted ultrasound data, it was determined that the probe can presently maintain intensity values up to 2.4 W/cm2 over indefinite times for therapeutic applications combined with intermittent 3D scanning to maintain targeting. These values were acquired using 8 cycle bursts at a prf of 6 kHz. Ex vivo heating experiments of excised pork tissue yielded a maximum temperature rises of 2.3 degrees C over 5 minutes of ultrasound exposure with an average rise of 1.8 +/- 0.2 degrees C over 5 trials. Modifications to the power supply and transducer array may enable us to reach the higher intensities required to facilitate drug delivery therapy.