Mutations and structural variants arising during double-strand break repair

Double-strand break (DSB) repair is highly mutagenic compared to normal replication. In budding yeast, repair of an HO endonuclease-induced DSB at MAT-alpha can be made with a transcriptionally silent HMR::Kl-URA3 donor. We isolated thousands of mutations occurring during this repair process. We conclude that the repair replication fork appears to have the two DNA strands open ~80 bp ahead of the DNA polymerase, but the strands re-anneal rapidly behind the polymerase. Additionally, we analyzed interchromosomal template switching, in which the partially copied DNA strand dissociates and pairs with a new template at a short stretch of perfectly matching bases (microhomology), and resumes copying. We show that these apparent microhomology-mediated template switching events in fact require the pairing of ~200 bp of imperfectly matching bases (homeology).