Damage-Seq: Mapping Single-Strand DNA Damage Hotspots

During a cell`s life, its DNA is constantly exposed to numerous exogenous and endogenous damaging agents. Damage accumulation on the DNA backbone or bases has cytotoxic and genotoxic effects, as it may lead to mutations, genomic instability and, consequently, cancer.

Although single-strand DNA (ss-DNA) damage does not prompt the same acute cell response caused by DNA double-strand breaks, different types of single-strand lesions are the most common form of damage incurred to the DNA molecule.

Deficiencies in DNA repair mechanisms may lead to severe pathogenic implications. It is therefore apparent that a technique for quantifying a cell`s DNA single-strand damage and repair state is of great value for both research and clinical applications. All of the methods currently available for the measurement of single-strand DNA damage are limited in that they address a specific type of damage and do not measure the overall damage state of the cell. Moreover, these methods do not provide information regarding the location of the damage along the genome.

In order to map ss-DNA damage hotspots along the genome we utilize a ChIP-seq based DNA pull-down method, based on a technique for labeling ss-DNA damage we have recently developed. Using a cocktail of viral and bacterial repair enzymes, ss-DNA lesions are excised from the double strand, leaving a single strand gap. Then, a repair synthesis reaction is performed using biotinylated nucleotides, leading to the incorporation of these nucleotides into the formerly damaged sites. DNA is then fragmented and immunoprecipitated using anti-biotin antibodies, resulting in a sample enriched with fragments that originally contained damaged nucleobases. After sequencing of the sample, reads are mapped back to the reference genome, and the coverage can be examined in order to identify hotspots.