Tracking break induced replication reveals its stalling at roadblocks

Break-induced replication (BIR) repairs one-ended double strand breaks (DSBs) similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human disease(1,2). Previous studies have defined the enzymes required for BIR(1–5); however, understanding of initial and extended BIR synthesis as well as how the migrating D-loop proceeds through known replication roadblocks has been precluded by technical limitations. Here, using a newly developed assay, we demonstrate that BIR synthesis initiates soon after strand invasion and proceeds slower than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but fails to proceed beyond 30 kb, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32 but does not proceed efficiently. We demonstrate that interstitial telomeric DNA disrupts and terminates BIR progression. Also, BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and on how BIR contributes to genome instability.