RESUMO
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.
Assuntos
Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Dano ao DNA , DNA de Cadeia Simples , Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação da Cromatina/métodos , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Replicação do DNA , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Ligação Proteica , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
A genome-wide deletion library is a powerful tool for probing gene functions and one has recently become available for the fission yeast Schizosaccharomyces pombe. Here we use deep sequencing to accurately characterize the barcode sequences in the deletion library, thus enabling the quantitative measurement of the fitness of fission yeast deletion strains by barcode sequencing.