Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution
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 metho...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2020-11-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/59111 |
| _version_ | 1811199299538649088 |
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| author | Shanaya Shital Shah Stella R Hartono Frédéric Chédin Wolf-Dietrich Heyer |
| author_facet | Shanaya Shital Shah Stella R Hartono Frédéric Chédin Wolf-Dietrich Heyer |
| author_sort | Shanaya Shital Shah |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T01:46:45Z |
| format | Article |
| id | doaj.art-0b49950c133f4c2382f5323ebaf1c5a8 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T01:46:45Z |
| publishDate | 2020-11-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-0b49950c133f4c2382f5323ebaf1c5a82022-12-22T03:53:03ZengeLife Sciences Publications LtdeLife2050-084X2020-11-01910.7554/eLife.59111Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distributionShanaya Shital Shah0https://orcid.org/0000-0002-2881-2794Stella R Hartono1Frédéric Chédin2Wolf-Dietrich Heyer3https://orcid.org/0000-0002-7774-1953Department of Microbiology and Molecular Genetics, University of California, Davis, Davis, United StatesDepartment of Molecular and Cellular Biology, University of California, Davis, Davis, United StatesDepartment of Molecular and Cellular Biology, University of California, Davis, Davis, United StatesDepartment of Microbiology and Molecular Genetics, University of California, Davis, Davis, United States; Department of Molecular and Cellular Biology, University of California, Davis, Davis, United StatesDisplacement 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.https://elifesciences.org/articles/59111recombinationgenome stabilityDNA repair |
| spellingShingle | Shanaya Shital Shah Stella R Hartono Frédéric Chédin Wolf-Dietrich Heyer Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution eLife recombination genome stability DNA repair |
| title | Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution |
| title_full | Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution |
| title_fullStr | Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution |
| title_full_unstemmed | Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution |
| title_short | Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution |
| title_sort | bisulfite treatment and single molecule real time sequencing reveal d loop length position and distribution |
| topic | recombination genome stability DNA repair |
| url | https://elifesciences.org/articles/59111 |
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