Investigating the properties of a repair replication‐fork in the budding yeast Saccharomyces cerevisiae

Abstract

Genome instability is the hallmark of several human diseases including cancer. The FoSTeS (Fork Stalling and Template Switching) model – also termed Microhomology‐Mediated Break‐induced Replication (MM‐BIR) – posits that the fork originating from a stalled or collapsed fork can trigger genome instability by means of template switching of the repair‐fork. In order to examine MM‐BIR in detail, we created a system in S. cerevisiae in which a single double‐strand break induced by HO endonuclease can be repaired by a two step process that involve 1) strand invasion with a donor sequence followed by 2) template switch located at about 50 kb away generating a functional URA3 gene as part of a complex nonreciprocal translocation. Our results show that when the sequences are 100 % homologous, these events occur at a frequency of about 2 × 10−3. The presence of sequence heterology had distinct affect on the two steps; heterology severely restricted strand invasion by about four orders of magnitude, while template switch was less affected. For the first time, our results reveal two important features distinguishing canonical strand invasions from template jumps: 1) template jumps were less restrained by heterologies than strand invasions providing evidence for their distinct nature and 2) invasions preferred the use of the very 3′ end whereas template jumps showed no preferred sequences, suggesting repeated strand dissociation and reformation as a hallmark of repair forks. Sequencing of junctions of recombined sequences that shared no significant homologies showed that such events utilized sequences ranging from 3 bp micro‐homologies to no homologies.