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Protocol for mapping double-stranded DNA break sites across the genome with translocation capture sequencing.

ABSTRACT: Translocation sequencing can be used to assess mechanisms of DNA repair and identify genome-wide double-strand breaks (DSBs) accessible to DNA repair machinery. Here, we present a protocol for mapping double-strand DNA break sites across the genome with translocation capture sequencing. Bait DSBs are introduced using a Cas9 nuclease and repaired by the host cell, connecting bait DSBs to other DSBs. Repair sites are detected by isolating bait site DNA, cleaving normal sequence to enrich off-site repair, and next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Switonski et al. (2021).¹.

SUBMITTER: Delaney JR

PROVIDER: S-EPMC10068614 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Protocol for mapping double-stranded DNA break sites across the genome with translocation capture sequencing.

Delaney Joe R JR La Spada Albert R AR

STAR protocols 20230330 2

Translocation sequencing can be used to assess mechanisms of DNA repair and identify genome-wide double-strand breaks (DSBs) accessible to DNA repair machinery. Here, we present a protocol for mapping double-strand DNA break sites across the genome with translocation capture sequencing. Bait DSBs are introduced using a Cas9 nuclease and repaired by the host cell, connecting bait DSBs to other DSBs. Repair sites are detected by isolating bait site DNA, cleaving normal sequence to enrich off-site ...[more]

PMID: 37000621

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Project description:During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Dataset Information

Protocol for mapping double-stranded DNA break sites across the genome with translocation capture sequencing.

Publications

Protocol for mapping double-stranded DNA break sites across the genome with translocation capture sequencing.

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