Low- and high-resolution mapping of DNA damage at specific sites

Measurement of DNA damage and repair at the nucleotide level in intact cell s has provided compelling evidence for the molecular details of these event s as they occur in intact organisms. Furthermore, these measurements give t he most accurate picture of the rates of repair in different structural dom ains of DNA in chromatin. In this report, we describe two methods currently used in our laboratories to map DNA lesions at (or near) nucleotide resolu tion in yeast cells. The low-resolution method couples damage-specific stra nd breaks in DNA with indirect end-labeling to measure DNA lesions over a s pan of 1.5 to 2 kb of DNA sequence. The resolution of this method is limite d by the resolution of DNA length measurements on alkaline agarose gels (ab out +/- 20 bp on average). The high-resolution method uses streptavidin mag netic beads and special biotinylated oligonucleotides to facilitate end-lab eling of DNA fragments specifically cleaved at damage sites. The latter met hod maps DNA damage sites at nucleotide resolution over a shorter distance (<500 bp), and is constrained to the length of DNA resolvable on DNA sequen cing gels. These methods are used in tandem for answering questions regardi ng DNA damage and repair in different chromatin domains and states of gene expression. (C) 2000 Academic Press.