S1-seq assay for mapping processed DNA ends Journal Article
| Authors: | Mimitou, E. P.; Keeney, S. |
| Article Title: | S1-seq assay for mapping processed DNA ends |
| Abstract: | During meiosis, the specialized cell division giving rise to gametes, numerous DNA double-strand breaks (DSBs) are introduced at multiple places throughout the genome by the topoisomerase-like protein Spo11. Homologous recombination, a highly conserved DSB repair pathway, is employed for their repair and ensures the formation of chiasmata and the proper segregation of homologous chromosomes. In the initial steps of recombination, end resection takes place, wherein Spo11 is endonucleolytically released and the 5′-terminal strands of each DSB are exonucleolytically processed, exposing the ssDNA necessary to identify a homologous repair template. DNA removed by DSB processing is reconstituted by DNA synthesis, which copies genetic information from the intact homologous template. We developed a next-generation sequencing assay, termed S1-seq, to study DNA end resection genome-wide at high-spatial resolution during yeast meiotic recombination. The assay relies on the fact that removal of the ssDNA tails of resected DSBs marks the position where resection stopped. Molecular features of resection are revealed by sequencing of these ssDNA-to-dsDNA junctions and comparison to high-resolution Spo11 DSB maps. We describe the experimental and computational methods for S1-seq as applied to meiosis in the SK1 strain of budding yeast Saccharomyces cerevisiae and discuss how it can also be applied to map DSBs and recombination intermediates. © 2018 Elsevier Inc. |
| Keywords: | nonhuman; polymerase chain reaction; meiosis; homologous recombination; assay; gel; double stranded dna; protein purification; saccharomyces cerevisiae; double stranded dna break; single stranded dna; dna double-strand breaks; bioinformatics; streptavidin; genomic dna; dna isolation; budding yeast; meiotic recombination; fungal strain; spo11; fungal cell culture; next generation sequencing; dna end resection; exo1; s1 seq assay |
| Journal Title: | Methods in Enzymology |
| Volume: | 601 |
| ISSN: | 0076-6879 |
| Publisher: | Academic Press |
| Date Published: | 2018-01-01 |
| Start Page: | 309 |
| End Page: | 330 |
| Language: | English |
| DOI: | 10.1016/bs.mie.2017.11.031 |
| PROVIDER: | scopus |
| PUBMED: | 29523237 |
| PMCID: | PMC5891823 |
| DOI/URL: | |
| Notes: | Chapter 13 in "Mechanisms of DNA Recombination and Genome Rearrangements: Intersection between Homologous Recombination, DNA Replication and DNA Repair" (ISBN: 978-0-12-813979-0) -- Export Date: 2 April 2018 -- Source: Scopus |
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