Biology subjects

Fishel, R.

Publications and source records attributed to Fishel, R..

7 recordsLinked to original sources

bioRxiv2025

A Python framework for magnetic tweezers real-time image processing and microscope control

Magnetic tweezers are a popular biophysical instrument for manipulating and measuring single molecules. Most groups rely on custom-built setups tailored to specific experiments, making it challenging to implement and share software. Typically, image acquisition and hardware control are automated via LabVIEW, while real-time video processing is implemented in C++/CUDA libraries. Live processing can eliminate the need to store raw video, enabling high throughput, fast acquisition rates, and simplified experimental workflows. However, no open-source general-purpose software framework currently unifies these capabilities for magnetic tweezers experiments. Here, we introduce MagTrack and MagScope open-source Python-based tools designed to fill this gap. MagTrack is an image-processing library that efficiently determines bead positions from magnetic-tweezers videos using CPU or GPU computation. MagScope is a comprehensive software framework offering a graphical user interface, real-time hardware control, data acquisition, and video processing. It is built on a multiprocessing architecture for responsive, high-throughput computation. Together, MagTrack and MagScope offer a fully customizable, end-to-end, open-source Python alternative to proprietary or fragmented systems, enabling laboratories to adapt and extend the framework according to their experimental needs.

biophysics↗

bioRxiv2024

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

biophysics↗

bioRxiv2021

The mouse mammary tumor virus intasome exhibits distinct dynamics on target DNA

Retroviral intasomes are complexes assembled from purified integrase (IN) and oligonucleotides mimicking viral DNA ends (vDNA). Recombinant intasomes faithfully recapitulate integration of vDNA into a target DNA. Structural studies of retroviral intasomes have revealed an array of IN oligomer forms, which appear to share a conserved intasome core coordinating the vDNA ends for strand transfer into the target DNA. Here we have explored the biochemical and dynamic properties of the mouse mammary tumor virus (MMTV) octameric intasome. We show that the MMTV intasome is remarkably stable compared to the prototype foamy virus (PFV) tetrameric intasome. MMTV integration activity peaks within the range of physiological ionic strength and is more active in the presence of manganese compared to magnesium. Single-molecule images demonstrate that the target DNA search by MMTV intasomes appears rate-limiting, similar to PFV intasomes. The time between strand transfer of the two MMTV vDNA ends into the target DNA is [~]3 fold slower than PFV intasomes. MMTV intasomes can form extremely stable, largely immobile filaments on a target DNA that are comprised of multiple intasomes. This unusual property suggests that MMTV intasomes may readily form higher order oligomers that might underpin their increased stability.

biochemistry↗

bioRxiv2021

DNA Breaks and Gaps Target Retroviral Integration

Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration.

biochemistry↗

bioRxiv2021

MutS and DNA Function as a Clamp Loader for the MutL Sliding Clamp During Mismatch Repair

DNA mismatch repair (MMR) is accomplished by highly conserved MutS and MutL homologs. MutS proteins recognize mismatch nucleotides and in the presence of ATP form a stable sliding clamp on the DNA. The MutS sliding clamp then promotes the cascade assembly of a MutL sliding clamp, which ultimately coordinates downstream mismatch excision. The MutS clamp-loader mechanics are unknown. Here we have examined a conserved positively charged cleft (PCC) located on the MutL N-terminal domain (NTD) proposed to mediate stable DNA binding events in several MMR models. We show that MutL does not bind DNA in physiological ionic conditions. Instead, the MutS sliding clamps and DNA together exploit the PCC to position the MutL NTD for clamp loading. Once in a sliding clamp form, the MutL PCC aids in UvrD helicase capture but not interactions with MutH during mismatch excision. The MutS-DNA clamp-loader progressions are significantly different from the replication clamp-loaders that attach polymerase processivity factors such as {beta}-clamp and PCNA to the DNA. These studies underlining the breadth of mechanisms for stably linking crucial genome maintenance proteins to the DNA.

biophysics↗

bioRxiv2021

The Formation of a Stable Sliding Clamp Discriminates MSH2-MSH3 and MSH2-MSH6 Mismatch Interaction

MutS homologs (MSH) are highly conserved core components of DNA mismatch repair (MMR). Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Once on DNA the MSH sliding clamps provide a platform for the assembly of MMR strand-specific excision components beginning with the highly conserved MutL homologs (MLH/PMS). Previous studies with short mismatch-containing oligonucleotides revealed an MSH ATP hydrolysis (ATPase) cycle that included mismatch recognition, the formation of an ATP-bound sliding clamp and dissociation from the end of a mismatched DNA that ultimately recovers the mismatch binding conformation. We found that ATP-bound MSH complexes on blocked-end or very long DNA are extremely stable under a range of ionic conditions. These observations underpinned the development of a high-throughput fluorescence resonance energy transfer (FRET) system capable of clearly distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 activities that is suitable for chemical inhibitor screens.

biochemistry↗

bioRxiv2020

REMBRANDT: A high-throughput barcoded sequencing approach for COVID-19 screening.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A still-debated percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions that includes diabetes, COPD and pregnancy. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. As communities re-open, large numbers of people will need to be tested and contact-tracing of positive patients will be required to prevent additional waves of infections and enable the continuous monitoring of the viral loads COVID-19 positive patients. A number of molecular assays are currently in clinical use to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening, a major bottleneck for testing samples from a large patient population for COVID-19.

molecular biology↗