scholarly journals Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

2020 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice ◽  
The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

scholarly journals Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella R Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Vitro Assay ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

scholarly journals Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

1993 ◽  
Vol 13 (4) ◽  
pp. 2162-2171 ◽  
Author(s):  
C S Madsen ◽  
S C Ghivizzani ◽  
W W Hauswirth
Keyword(s):  
Mitochondrial Dna ◽  
Protein Binding ◽  
Loop Formation ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Loop Region ◽  
In Organello ◽  
Close Proximity ◽  
D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

scholarly journals Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

2021 ◽  
Author(s):  
Terren Chang ◽  
Xi Long ◽  
Shankar Shastry ◽  
Joseph William Parks ◽  
Michael D Stone
Keyword(s):  
Single Molecule ◽  
Mechanical Analysis ◽  
Magnetic Tweezers ◽  
Repeat Sequence ◽  
Single Stranded Dna ◽  
Telomere Repeat ◽  
D Loop ◽  
Human Telomere ◽  
Strand Invasion

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

scholarly journals 5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

2020 ◽  
Author(s):  
Grace Mullally ◽  
Kara van Aelst ◽  
Mohsin M. Naqvi ◽  
Fiona M. Diffin ◽  
Tautvydas Karvelis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Rna Aptamers ◽  
Loop Formation ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Rnp Complex ◽  
Target Binding ◽  
The Impact

A key aim in exploiting CRISPR-Cas is the engineering of gRNA to introduce additional functionalities, ranging from small nucleotide changes that increase efficiency of on-target binding to the inclusion of large functional RNA aptamers and ribonucleoproteins (RNPs. Interactions between gRNA and Cas9 are crucial for RNP complex assembly but several distinct regions of the gRNA are amenable to modification. Using a library of modified gRNAs, we used in vitro ensemble and single-molecule assays to assess the impact of RNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that R-loop formation and DNA cleavage activity are essentially unaffected by gRNA modifications of the Upper Stem, first Hairpin and 3’ end. In contrast, 5’ additions of only two or three nucleotides reduced R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such gRNA modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5’ end supported formation of a stable ~9 bp R-loop that could not activate DNA cleavage. These observations will assist in successful gRNA design.

Single Molecule Force Spectroscopy Maps to Study Receptors Clustering

2001 ◽  
Vol 7 (S2) ◽  
pp. 860-861
Author(s):  
R. Bhatia ◽  
N. Almqvist ◽  
S. Banerjee ◽  
G. Primbs ◽  
N. Desai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Single Molecule ◽  
Regional Distribution ◽  
Cellular Membrane ◽  
Angiogenic Factor ◽  
Extracellular Region ◽  
Force Volume ◽  
Endothelial Growth Factor

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).

Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region

1993 ◽  
Vol 13 (4) ◽  
pp. 2162-2171
Author(s):  
C S Madsen ◽  
S C Ghivizzani ◽  
W W Hauswirth
Keyword(s):  
Mitochondrial Dna ◽  
Protein Binding ◽  
Loop Formation ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Loop Region ◽  
In Organello ◽  
Close Proximity ◽  
D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing

2009 ◽  
Vol 37 (2) ◽  
pp. 427-432 ◽  
Author(s):  
Jin Li ◽  
G. Mike Makrigiorgos
Keyword(s):  
Genetic Testing ◽  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
Mutation Detection ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Wild Type ◽  
Variant Alleles

PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and Makrigiorgos (2008) Nat. Med. 14, 579–584], a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost all genetic analysis, COLD-PCR provides a general platform to improve the sensitivity of essentially all DNA-variation detection technologies including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR combined with real-time PCR provides a new approach to boost the capabilities of existing real-time mutation detection methods. We replaced regular PCR with COLD-PCR before sequencing or real-time mutation detection assays to improve mutation detection-sensitivity by up to 100-fold and identified novel p53/Kras/EGFR (epidermal growth factor receptor) mutations in heterogeneous cancer samples that were missed by all existing methods. For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, DNA methylation testing and prenatal identification of fetal alleles in maternal blood.

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