Complex repeat structure promotes hyper-amplification and amplicon evolution through rolling-circle replication

Inverted repeats (IRs) are abundant in genomes and frequently serve as substrates for chromosomal aberrations, including gene amplification. In the early stage of amplification, repeated cycles of chromosome breakage and rearrangement, called breakage-fusion-bridge (BFB), generate a large inverted structure, which evolves into highly-amplified, complex end products. However, it remains to be determined how IRs mediate chromosome rearrangements and promote subsequent hyper-amplification and amplicon evolutions. To dissect the complex processes, we constructed repetitive structures in a yeast chromosome and selected amplified cells using genetic markers with limited expression. The genomic architecture was associated with replication stress and produced extra-/intra-chromosomal amplification. Genetic analysis revealed structure-specific endonucleases, Mus81 and Rad27, and post-replication DNA repair protein, Rad18, suppress the amplification processes. Following BFB cycles, the intra-chromosomal products undergo intensive rearrangements, such as frequent inversions and deletions, indicative of rolling-circle replication. This study presents an integrated view linking BFB cycles to hyper-amplification driven by rolling-circle replication.