Graduate Program, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Genome Res. 2013 Apr;23(4):705-15. doi: 10.1101/gr.146357.112. Epub 2012 Dec 17.
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.
自发的 DNA 损伤可能在基因组中非随机发生，特别是当基因组维护机制受到破坏时。我们开发了单链 DNA（ssDNA）相关蛋白免疫沉淀测序（SPI-seq）方法，以绘制基因组 DNA 损伤热点图。我们用 Rad52 同源重组修复蛋白来证明这种方法，Rad52 可以结合在 DNA 损伤处形成的 ssDNA。SPI-seq 忠实地检测到在裂殖酵母中，Rad52 在人为诱导的双链断裂（DSBs）以及内源性编程的交配型转换 DSB 处的富集。将 Rad52 SPI-seq 应用于裂殖酵母 Pfh1 解旋酶或组蛋白 H3K56 去乙酰化酶 Hst4 突变体，导致在这些突变体中出现 DNA 损伤热点的全局视图。我们还偶然发现，组蛋白剂量异常会激活反转录转座子 Tf2，并导致 Rad52 结合的 Tf2 cDNA 物种的积累。SPI-seq 应该广泛适用于绘制 DNA 损伤位点图，并揭示基因组不稳定性的原因。
