Re-Replication Induced Gene Amplification: Phenomenon, Mechanism, and Significance

Eukaryotic cells employ a battery of overlapping control mechanisms to ensure that each segment of their genome is replicated once, and only once, per cell cycle. While the long standing view of the field is that this tight block to re-replication is necessary for the preservation of genome integrity, there is no direct experimental evidence supporting this belief. The work presented in Chapter 2 critically evaluates this idea and demonstrates that experimental induction of low, sub-lethal levels of re-replication in Saccharomyces cerevisiae can indeed cause at least one form of genomic instability, namely gene amplification. The mechanism of such Re-Replication Induced Gene Amplification (RRIGA) is explored in Chapter 3. There, I show that re-replication forks are prone to frequent breakage, which instigates a repair response. Non-allelic repetitive sequence elements positioned to either side of the re-initiating origin undergo homologous recombination through a single-stranded annealing mechanism, ultimately generating a head-to-tail duplication in loco. These duplications have repetitive sequence elements at the amplicon boundaries and a hybrid repetitive element at the inter-amplicon junction. The details of the mechanism provide insight into why re-replication is so efficient at producing segmental amplifications. Finally, in Chapter 4 I address the relevance of RRIGA. There I present evidence that RRIGA is a likely driver of spontaneous segmental amplifications in cells with intact replication controls. Furthermore, I show that very slight disruption of replication control greatly increases the rate of such amplifications. These findings strongly suggest a role for RRIGA in both evolution and oncogenesis