scholarly journals Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

2019 ◽  
Author(s):  
Sungchul Kim ◽  
Luuk Loeff ◽  
Sabina Colombo ◽  
Stan J.J. Brouns ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Dependent Manner ◽  
Dna Fragments ◽  
Comprehensive Understanding ◽  
Single Molecule Fluorescence ◽  
Spatiotemporal Resolution ◽  
Single Stranded Dna ◽  
Correct Orientation ◽  
Crispr Array ◽  
Cellular Dna

AbstractCRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including single-stranded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1-Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.

scholarly journals Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

2019 ◽  
Author(s):  
Anna A. Shiriaeva ◽  
Ekaterina Savitskaya ◽  
Kirill A. Datsenko ◽  
Irina O. Vvedenskaya ◽  
Iana Fedorova ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Type I ◽  
Dependent Manner ◽  
Dna Fragments ◽  
Crispr Array ◽  
Protospacer Adjacent Motif ◽  
Sequencing Method ◽  
Foreign Dna ◽  
And Function

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves “adaptation,” the integration of ~30-bp DNA fragments into the CRISPR array as “spacer” sequences, and “interference,” the targeted degradation of DNA containing a “protospacer” sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1–4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with “primed adaptation,” ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas “self-targeting” locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, “FragSeq,” and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.

scholarly journals Homologous Recombination under the Single-Molecule Fluorescence Microscope

2019 ◽  
Vol 20 (23) ◽  
pp. 6102
Author(s):  
Dalton R. Gibbs ◽  
Soma Dhakal
Keyword(s):  
Homologous Recombination ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Dna Interactions ◽  
Single Molecule Fluorescence ◽  
Spatiotemporal Resolution ◽  
Dynamic Interactions ◽  
Protein Dna Interactions ◽  
Dna Substrates ◽  
Single Molecule Fluorescence Microscopy

Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein–protein and protein–DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination.

scholarly journals Modelling single-molecule kinetics of helicase translocation using high-resolution nanopore tweezers (SPRNT)

2021 ◽  
Author(s):  
Jonathan M. Craig ◽  
Andrew H. Laszlo ◽  
Ian C. Nova ◽  
Jens H. Gundlach
Keyword(s):  
Single Molecule ◽  
Dwell Time ◽  
Specific Enzyme ◽  
Spatiotemporal Resolution ◽  
Single Stranded Dna ◽  
Detailed Model ◽  
Specific Location ◽  
Dna Strand ◽  
Kinetics Of ◽  
Dna Substrate

Abstract Single-molecule picometer resolution nanopore tweezers (SPRNT) is a technique for monitoring the motion of individual enzymes along a nucleic acid template at unprecedented spatiotemporal resolution. We review the development of SPRNT and the application of single-molecule kinetics theory to SPRNT data to develop a detailed model of helicase motion along a single-stranded DNA substrate. In this review, we present three examples of questions SPRNT can answer in the context of the Superfamily 2 helicase Hel308. With Hel308, SPRNT’s spatiotemporal resolution enables resolution of two distinct enzymatic substates, one which is dependent upon ATP concentration and one which is ATP independent. By analyzing dwell-time distributions and helicase back-stepping, we show, in detail, how SPRNT can be used to determine the nature of these observed steps. We use dwell-time distributions to discern between three different possible models of helicase backstepping. We conclude by using SPRNT’s ability to discern an enzyme’s nucleotide-specific location along a DNA strand to understand the nature of sequence-specific enzyme kinetics and show that the sequence within the helicase itself affects both step dwell-time and backstepping probability while translocating on single-stranded DNA.

Optimization of Single-Molecule Fluorescence Burst Detection of ds-DNA: Application to Capillary Electrophoresis Separations of 100–1000 Basepair Fragments

1997 ◽  
Vol 51 (10) ◽  
pp. 1579-1584 ◽  
Author(s):  
Brian B. Haab ◽  
Richard A. Mathies
Keyword(s):  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Laser Power ◽  
Detection Efficiency ◽  
Fragment Size ◽  
Signal To Noise Ratio ◽  
Burst Size ◽  
Dna Fragments ◽  
Burst Detection ◽  
Single Molecule Fluorescence

Methods for optimizing the dye labeling, laser excitation, and data analysis for single-molecule fluorescence burst detection of ds-DNA have been developed and then validated through capillary electrophoresis (CE) separations of 100–1000 basepair (bp) DNA. Confocal microscopy is used to observe fluorescence bursts from individual DNA fragments labeled with the intercalation dye TO6 as they pass through the ∼ 2-μm-diameter focused laser beam. The dye concentration and laser power were optimized by studying fluorescence burst intensities from pBluescript DNA fragments. The optimal TO6 concentration was ≤100 nM, and the optimal laser power was ≤1 mW. Single-molecule counting was then used to detect CE separations of a 100–1000 bp DNA sizing ladder in 3% linear polyacrylamide. Discrete and baseline-resolved fluorescence bursts were observed in bands as small as 100 bp, and the average burst size within each band increased linearly with fragment size. By counting events using a single optimally chosen discriminator level, we achieve maximum signal-to-noise ratio (S/N) for each fragment size. If the discriminator level is ramped linearly with fragment size to achieve a constant detection efficiency, then the number of events properly reflects the relative fragment concentrations.

Dispersion Correction Alleviates Dye Stacking of Single-Stranded DNA and RNA in Simulations of Single-Molecule Fluorescence Experiments

2018 ◽  
Vol 122 (49) ◽  
pp. 11626-11639 ◽  
Author(s):  
Kara K. Grotz ◽  
Mark F. Nueesch ◽  
Erik D. Holmstrom ◽  
Marcel Heinz ◽  
Lukas S. Stelzl ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dispersion Correction ◽  
Single Molecule Fluorescence ◽  
Single Stranded Dna ◽  
Dna And Rna

scholarly journals Direct Single-Molecule Observation of Sequential DNA Bending Transitions by the Sox2 HMG Box

2018 ◽  
Vol 19 (12) ◽  
pp. 3865 ◽  
Author(s):  
Mahdi Moosa ◽  
Phoebe Tsoi ◽  
Kyoung-Jae Choi ◽  
Allan Ferreon ◽  
Josephine Ferreon
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Single Molecule ◽  
Dna Bending ◽  
Differential Regulation ◽  
Dependent Manner ◽  
Biological Functions ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Pioneer Transcription Factor

Sox2 is a pioneer transcription factor that initiates cell fate reprogramming through locus-specific differential regulation. Mechanistically, it was assumed that Sox2 achieves its regulatory diversity via heterodimerization with partner transcription factors. Here, utilizing single-molecule fluorescence spectroscopy, we show that Sox2 alone can modulate DNA structural landscape in a dosage-dependent manner. We propose that such stoichiometric tuning of regulatory DNAs is crucial to the diverse biological functions of Sox2, and represents a generic mechanism of conferring functional plasticity and multiplicity to transcription factors.

scholarly journals RecA filament sliding on DNA facilitates homology search

eLife ◽  
2012 ◽  
Vol 1 ◽  
Author(s):  
Kaushik Ragunathan ◽  
Cheng Liu ◽  
Taekjip Ha
Keyword(s):  
Diffusion Coefficient ◽  
Single Molecule ◽  
Homology Search ◽  
Single Molecule Fluorescence ◽  
Base Pairs ◽  
Spatiotemporal Resolution ◽  
Present Evidence ◽  
Encounter Complex ◽  
Homology Recognition ◽  
Recognition Efficiency

During homologous recombination, RecA forms a helical filament on a single stranded (ss) DNA that searches for a homologous double stranded (ds) DNA and catalyzes the exchange of complementary base pairs to form a new heteroduplex. Using single molecule fluorescence imaging tools with high spatiotemporal resolution we characterized the encounter complex between the RecA filament and dsDNA. We present evidence in support of the ‘sliding model’ wherein a RecA filament diffuses along a dsDNA track. We further show that homology can be detected during sliding. Sliding occurs with a diffusion coefficient of approximately 8000 bp2/s allowing the filament to sample several hundred base pairs before dissociation. Modeling suggests that sliding can accelerate homology search by as much as 200 fold. Homology recognition can occur for as few as 6 nt of complementary basepairs with the recognition efficiency increasing for higher complementarity. Our data represents the first example of a DNA bound multi-protein complex which can slide along another DNA to facilitate target search.

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