CONSTRUCTION OF A VECTOR CONTAINING A SITE-SPECIFIC DNA DOUBLE-STRANDBREAK WITH 3'-PHOSPHOGLYCOLATE TERMINI AND ANALYSIS OF THE PRODUCTS OF END-JOINING IN CV-1 CELLS

Previous studies have shown that linearized SV40-based shuttle vectors transfected into mammalian cells are efficiently recircularized by an error-prone end-joining pathway. To determine whether and with what s pecificity free radical-mediated double-strand breaks are rejoined by this pathway, a structural mimic of such a break was introduced at a s pecific site in an SV40-based shuttle vector, by ligating purified 3'- phosphoglycolate-terminated oligonucleotides into 3' recessed ends gen erated in the linearized vector. These terminally blocked linear vecto rs were efficiently repaired and replicated when transfected into simi an CV-1 cells. Sequencing across the repair joints in progeny plasmid indicated that, for a blunt-ended vector, the most frequent mechanism of rejoining was splicing at a terminal 4-base homology; however, a si gnificant fraction of the joints retained all bases from both ends of the break, consistent with a mechanism involving simple 3'-phosphoglyc olate removal, followed by blunt-end ligation. For the analogous 3'-hy droxyl terminated break, the fraction of simple blunt-end ligations wa s considerably higher. For a phosphoglycolate-terminated vector with c ohesive ends the most frequent repair mechanism was simple ligation of the annealed cohesive ends, presumably preceded by phosphoglycolate r emoval. For all these substrates, the remaining repair joints showed s mall or large deletions from one or both of the ends, usually with app arent annealing at short (1-4-base) homologies. The results suggest th at while breaks with 3'-phosphoglycolates can be repaired, these block ed termini represent a significant barrier to DNA end-joining, and can significantly alter its specificity The presence of cohesive ends app ears to improve markedly the fidelity of rejoining for terminally bloc ked double-strand breaks.