scholarly journals Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase

2009 ◽  
Vol 35 (5) ◽  
pp. 694-703 ◽  
Author(s):  
Masayoshi Honda ◽  
Jeehae Park ◽  
Robert A. Pugh ◽  
Taekjip Ha ◽  
Maria Spies
Keyword(s):  
Single Molecule ◽  
Differential Effect ◽  
Binding Proteins ◽  
Dna Translocation ◽  
Ssdna Binding ◽  
Single Molecule Analysis ◽  
Ssdna Binding Proteins ◽  
Xpd Helicase

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 Single-Molecule Analysis of Conformational Transitions in XPD Helicase

2013 ◽  
Vol 104 (2) ◽  
pp. 61a
Author(s):  
Mohamed K. Ghoneim ◽  
Maria Spies
Keyword(s):  
Single Molecule ◽  
Conformational Transitions ◽  
Single Molecule Analysis ◽  
Xpd Helicase

scholarly journals Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

2019 ◽  
Author(s):  
O.J. Wilkinson ◽  
C. Carrasco ◽  
C. Aicart-Ramos ◽  
F. Moreno-Herrero ◽  
M.S. Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

ABSTRACTDNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5’ to 3’ ssDNA translocase and 5’ to 3’ helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ∼200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

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.

Novel homologs of replication protein A in Archaea: implications for the evolution of ssDNA-binding proteins

1998 ◽  
Vol 23 (8) ◽  
pp. 273-277 ◽  
Author(s):  
Frédéric Chédin ◽  
Erica M Seitz ◽  
Stephen C Kowalczykowski
Keyword(s):  
Binding Proteins ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Ssdna Binding ◽  
Ssdna Binding Proteins

scholarly journals Bulk and single-molecule analysis of a bacterial DNA2-like helicase–nuclease reveals a single-stranded DNA looping motor

2020 ◽  
Vol 48 (14) ◽  
pp. 7991-8005
Author(s):  
Oliver J Wilkinson ◽  
Carolina Carrasco ◽  
Clara Aicart-Ramos ◽  
Fernando Moreno-Herrero ◽  
Mark S Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

Abstract DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase–nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5′ to 3′ ssDNA translocase and 5′ to 3′ helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase–nucleases and DNA looping motor proteins in general.

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.

scholarly journals A meiosis-specific factor C19orf57/4930432K21Rik/BRME1 modulates localization of RAD51 and DMC1 recombinases to DSBs in mouse meiotic recombination

2020 ◽  
Author(s):  
Kazumasa Takemoto ◽  
Naoki Tani ◽  
Yuki Takada-Horisawa ◽  
Sayoko Fujimura ◽  
Nobuhiro Tanno ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Meiotic Recombination ◽  
Binding Proteins ◽  
Strand Break ◽  
Specific Factor ◽  
Dsb Repair ◽  
Ssdna Binding ◽  
Dna Double Strand Break ◽  
Multiple Processes ◽  
Ssdna Binding Proteins

SummaryMeiotic recombination is critical for genetic exchange and generation of chiasmata that ensures faithful chromosome segregation during meiosis I. Meiotic recombination is initiated by DNA double-strand break (DSB) followed by multiple processes of DNA repair. The exact mechanisms how recombinases localize to DSB remained elusive. Here we show that C19orf57/4930432K21Rik/BRME1 is a new player for meiotic recombination in mice. C19orf57/4930432K21Rik/BRME1 associates with ssDNA binding proteins, BRCA2 and MEILB2/HSF2BP, critical recruiters of recombinases onto DSB sites. Disruption of C19orf57/4930432K21Rik/BRME1 shows severe impact on DSB repair and male fertility. Remarkably, removal of single stranded DNA (ssDNA) binding proteins from DSB sites is delayed, and reciprocally the loading of RAD51 and DMC1 onto resected ssDNA is impaired in Brme1 KO spermatocytes. We propose that C19orf57/4930432K21Rik/BRME1 modulates localization of recombinases to meiotic DSB sites through the interaction with the BRCA2-MEILB2/HSF2BP complex during meiotic recombination.

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