Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

Konomura, Naoto; Arai, Naoto; Shinohara, Tokuko; Kobayashi, Jun; Iwasaki, W.; Ikawa, Shiro; Kusano, Kohji; Shibata, Takehiko

doi:10.1093/nar/gkw998

Cited by 6 publications

(6 citation statements)

References 65 publications

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“…In our datasets, the rad51-Δ and rfa1-S373P strains showed some similarities, including modest increases in short-range inversions but no increases in long-range inversions, when compared to the WT under CPT + NOC stress (Fig 4). Under CPT + αF stress, the rad51-Δ strain presented the same pattern as the dnl4-Δ, yku70-Δ and mre11-Δ strains, which would support previously-reported roles in end-processing for Mre11 [26] and Rad51 [27].…”

Section: Resultssupporting

confidence: 87%

Short-range, orientation-reversing template-switching events occur at a high frequency in the human and yeast genomes

Tremblay‐Belzile

Giorgio

Loubert‐Hudon

et al. 2020

Preprint

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20The identification of structural variations in genomes using next-generation sequencing approaches 21 greatly facilitates the study of genetic and genomic diseases. The data generated using these 22 approaches also provide interesting new means to examine DNA repair, recombination, and 23 replication to better understand sources of genomic instability. To better utilize this data, we 24 developed SCARR (Systematic Combination of Alignments to Recreate Rearrangements) to identify 25 DNA rearrangements, and used it to examine the occurrence of orientation-reversing events in human 26 and budding yeast genomes. SCARR exceeds the sensitivity of previous genome sequencing 27 approaches, and identifies rearrangements genome-wide with base-pair resolution, which helps 28 provide insights into the mechanisms involved in their formation. We find that short-range orientation-29 reversing events occur at high rates in both human and yeast genomes. We quantified these 30 rearrangements in yeast strains lacking various DNA repair factors, and propose that these short-31 range events often occur through template-switching events within a replication fork. We hypothesize 32 that this mechanism may act as an error-prone alternative to fork reversal to restart stalled replication 33 forks. 343 57 mitochondria using next-generation sequencing [11]. This type of approach revealed that these 58 events in organelles are also favored by sequence similarities as short as 5 bases, and may even 59 occur in the absence of homology. The circumstances leading to their formation, as well as their 60 possible consequences, remain poorly understood due to the challenges involved in their detection by 61 traditional biochemical methods [12]. 62The rapidly expanding repositories of next-generation sequencing data have opened several new 63 avenues for understanding genomic instability including its roles in evolution and genomic diseases. A 64 landmark study in the field identified tens of thousands of SVs within the human genome [2], which 65 included deletions, duplications, insertions, as well as a small number of inversions. New 66 computational approaches to detect and investigate SVs continue to be developed to improve 67 detection rates and focus on specific types of SVs, such as the harder to detect inversions [13][14][15]. 68Nevertheless, complex SVs such as those that result from FoSTeS and other template-switching 69 events often remain undetected by current next-generation sequencing analysis pipelines. 70To avoid a bias for simple SVs, we developed SCARR (Systematic Combination of Alignments to 71 Recreate Rearrangements), which uses no a priori assumptions about the relative positions of DNA 72 sequences around a breakpoint. SCARR instead iterates through all possible genome-wide alignment 73 combinations to find the best match. Using this approach, it is possible to identify deletions, tandem 74 duplications and breakpoints resulting from template-switching events at base-pair resolution with 75 reasonable accuracy. Consequently...

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Section: Resultssupporting

confidence: 87%

Short-range, orientation-reversing template-switching events occur at a high frequency in the human and yeast genomes

Tremblay‐Belzile

Giorgio

Loubert‐Hudon

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Following a rate-limiting nucleation step by a cluster of 2-6 RecA monomers, filaments are rapidly extended by first-order reaction kinetics by sequential monomer addition 43 . In the presence of ATPγs, saturation of the oligonucleotides with RecA results in juxta-position of neighbouring filaments, resulting in concatenation [44][45][46] and the appearance of longer filaments than would be expected from the contour length of a single oligonucleotide fully decorated with RecA. Moreover, the length of filaments on cryo-electron micrographs is easily quantifiable with basic image analysis tools.…”

Section: Trapping Pre-steady State Kinetic Intermediates By Cryo-emmentioning

confidence: 99%

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mäeots

Lee

Nans

et al. 2020

Preprint

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Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise shortlived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

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“…Following a rate-limiting nucleation step by a cluster of 2–6 RecA monomers, filaments are rapidly extended by first-order reaction kinetics by sequential monomer addition 44 . In the presence of ATPγS, saturation of the oligonucleotides with RecA results in juxta-position of neighbouring filaments, resulting in concatenation 45 – 47 and the appearance of longer filaments than would be expected from the contour length of a single oligonucleotide fully decorated with RecA. Moreover, the length of filaments on cryo-electron micrographs is easily quantifiable with basic image analysis tools.…”

Section: Resultsmentioning

confidence: 99%

Modular microfluidics enables kinetic insight from time-resolved cryo-EM

et al. 2020

View full text Add to dashboard Cite

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.

show abstract

Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

Cited by 6 publications

References 65 publications

Short-range, orientation-reversing template-switching events occur at a high frequency in the human and yeast genomes

Short-range, orientation-reversing template-switching events occur at a high frequency in the human and yeast genomes

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Modular microfluidics enables kinetic insight from time-resolved cryo-EM

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