scholarly journals Assessing protein dynamics on low complexity single-strand DNA curtains

2018 ◽  
Author(s):  
Jeffrey M. Schaub ◽  
Hongshan Zhang ◽  
Michael M. Soniat ◽  
Ilya J. Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
High Throughput ◽  
Single Molecule ◽  
Binding Proteins ◽  
Low Complexity ◽  
Rolling Circle Replication ◽  
Sequence Composition ◽  
Ssdna Binding ◽  
Length Changes ◽  
Ssdna Binding Proteins

AbstractSingle-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. As ssDNA is more flexible than double-stranded (ds)DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription and repair.

scholarly journals Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jason C Bell ◽  
Bian Liu ◽  
Stephen C Kowalczykowski
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Intramolecular Interactions ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Intramolecular Condensation ◽  
Nucleoprotein Complexes ◽  
Ssdna Binding Proteins ◽  
Dna Maintenance

Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.

scholarly journals Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

2006 ◽  
Vol 27 (5) ◽  
pp. 1592-1601 ◽  
Author(s):  
Naduparambil K. Jacob ◽  
Rachel Lescasse ◽  
Benjamin R. Linger ◽  
Carolyn M. Price
Keyword(s):  
Telomere Length ◽  
Binding Proteins ◽  
Growth Arrest ◽  
Cell Cycle Checkpoint ◽  
Cell Phenotype ◽  
Ssdna Binding ◽  
Cycle Checkpoint ◽  
Length Regulation ◽  
A Cell ◽  
Ssdna Binding Proteins

ABSTRACT The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3′ strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.

scholarly journals Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

2016 ◽  
Vol 113 (44) ◽  
pp. E6749-E6756 ◽  
Author(s):  
P. Benjamin Stranges ◽  
Mirkó Palla ◽  
Sergey Kalachikov ◽  
Jeff Nivala ◽  
Michael Dorwart ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Dna Synthesis ◽  
Lipid Bilayers ◽  
High Throughput ◽  
Single Molecule ◽  
Low Cost ◽  
Electrode Array ◽  
Oxide Semiconductor ◽  
Sequencing Platform ◽  
Sequencing By Synthesis

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.

scholarly journals A-type lamins are critical for the recruitment of RPA and RAD51 to stalled replication forks to maintain fork stability

2021 ◽  
Author(s):  
Simona Graziano ◽  
Nuria Coll-Bonfill ◽  
Barbara Teodoro-Castro ◽  
Sahiti Kuppa ◽  
Jessica Jackson ◽  
...  
Keyword(s):  
Replication Fork ◽  
Genome Stability ◽  
Binding Proteins ◽  
Physical Interaction ◽  
Ssdna Binding ◽  
Point Of Entry ◽  
Recombination Repair ◽  
Dna Strands ◽  
Increased Sensitivity ◽  
Ssdna Binding Proteins

Lamins provide a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. The mechanisms whereby lamins regulate genome stability remain poorly understood. Here, we demonstrate a crucial role for A-type lamins preserving the integrity of the replication fork (RF) during replication stress (RS). We find that lamins bind to nascent DNA strands, especially during RS, and ensure the recruitment of fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, play crucial roles in the stabilization, remodeling and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamins depletion elicits replication fork instability (RFI) depicted by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced accumulation of DNA damage, and increased sensitivity to replication inhibitors. Importantly, in contrast to cells deficient in various homology recombination repair proteins, the RFI phenotype of lamins-depleted cells is not linked to RF reversal. This suggests that the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamins-depleted cells is rescued by forced elevation of the heterotrimeric RPA complex or RAD51. These data unveil a clear involvement of structural nuclear proteins in the protection of ssDNA from the action of nucleases during RS by warranting proper recruitment of ssDNA binding proteins RPA and RAD51 to stalled RFs. In support of this model, we show physical interaction between RPA and lamins. Our study also suggests that RS is a major source of genomic instability in laminopathies and in lamins-depleted tumors.

High‐throughput single‐molecule imaging system using nanofabricated trenches and fluorescent DNA‐binding proteins

2020 ◽  
Vol 117 (6) ◽  
pp. 1640-1648
Author(s):  
Yujin Kang ◽  
Na Young Cheon ◽  
Jongjin Cha ◽  
Ayoung Kim ◽  
Hyung‐il Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
High Throughput ◽  
Single Molecule ◽  
Binding Proteins ◽  
Imaging System ◽  
Dna Binding Proteins ◽  
Single Molecule Imaging

scholarly journals RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Vol 48 (14) ◽  
pp. 7834-7843
Author(s):  
Hongshan Zhang ◽  
Jeffrey M Schaub ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Negative Regulator ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

Abstract RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

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