Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Abstract

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)-associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.

Dicer Promotes Transcription Termination at Sites of Replication Stress to Maintain Genome Stability

“…Indeed we have previously observed a substantial increase in Rad52 foci in dcr1 Δ cells as compared to WT (Zaratiegui et al, 2011), however the total level of Rad52 remains unchanged (data not shown), indicating that the Rad52 pool is limited. Rad52 nucleation occurs at sites of DNA damage during S-phase and subsequently spreads from the stall site (Zhou et al, 2013). To determine the precise location of replication stalling within rDNA we synchronized cells and performed Rad52 ChIP in S-phase.…”

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing

“…1b). After washing, bead-bound ssDNAs are unidirectionally ligated to a bridge adapter 18 . Adapter-ligated, bead-bound ssDNA fragments are then subjected to nested PCR to incorporate a barcode sequence necessary for de-multiplexing (Fig.…”

“…This improved method - LAM-HTGTS 6 - incorporates LAM-PCR 16,17 , bridge adapter ligation 18 , and a customized algorithm to fully characterize sequence reads with respect to the bait DSB employed. LAM-PCR employs a single primer to directly amplify bait-prey junctions from genomic DNA and generate single-stranded DNA (ssDNA) products with diverse ends 16,17 (Fig.…”

“…1b). The bridge adapter, with a short and nucleotide-variable 3’ overhang, provides a hybridized dsDNA “bridge” to stabilize ligation of the adapter to the diverse ssDNA ends using T4 DNA ligase 18 (Fig. 1b).…”

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

“…Mutations of both HNH nuclease and RuvC catalytic domains (DM-Cas9-CV) abolished the cleavage activity (Supplementary information, Figure S1A, lane5). We performed chromatin immunoprecipitation using high affinity nanobody for the Venus protein (GBP) [13]. Cas9-CV was significantly enriched in the emx1 locus but not control egfa-t1 locus in an sgRNA-dependent manner, while DM-Cas9-CV showed a greatly enhanced binding in comparison with Cas9-CV ( Figure 1B and Supplementary information, Figure S1E).…”