Physical Readouts of DNA Single Strand Damage

Hila Sharim, Chemical Physics, Tel Aviv University, Tel Aviv, Israel

DNA damage and repair are linked to fundamental biological processes such as metabolism, disease and aging. Deficiencies in DNA repair mechanisms have been shown to lead to severe pathogenic implications. Although single-strand lesions are the most abundant form of DNA damage, most existing techniques for quantifying these types of damage are lacking. We propose two methods for characterizing DNA single-strand damage. The first is a single molecule approach which takes advantage of natural repair enzymes to label damage sites in vitro by incorporating fluorescent nucleotides as a part of the repair process. The labeled DNA is extended on a microscope slide and damage sites are visualized as fluorescent spots along the DNA contour. Using this method, it is possible to quantify the extent of single-strand damage in the cell, as well as follow repair dynamics and efficiency. We next present a ChIP-seq based DNA pull-down method for targeting specific DNA regions, in order to map damage "hotspots" along the genome. Biotinylated nucleotides are incorporated into the DNA, which is then fragmented and immunoprecipitated using anti-biotin antibodies. These fragments are then sequenced and mapped to a reference genome, and sequencing coverage is examined to identify damage "hotspots" along the genome.