Segmental Duplications Arise from Pol32-Dependent Repair of Broken Forks through Two Alternative Replication-Based Mechanisms

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Abstract
The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Polδ is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions. Duplications of long segments of chromosomes are frequently observed in multicellular organisms (∼5% of our genome, for instance). They appear as a fundamental trait of the recent genome evolution in great apes and are often associated with chromosomal instability, capable of increasing genetic polymorphism among individuals, but also having dramatic consequences as a source of diseases and cancer. Despite their importance, the molecular mechanisms of formation of segmental duplications remain unclear. Using a specifically designed experimental system in the baker's yeast Saccharomyces cerevisiae, hundreds of naturally occurring segmental duplications encompassing dozens of genes were selected. With the help of modern molecular methods coupled to detailed genetic analysis, we show that such duplication events are frequent and result from untimely DNA synthesis accidents produced by two distinct molecular mechanisms: the well-known break-induced replication and a novel mechanism of template switching between low-complexity or microhomologous sequences. These two mechanisms, rather than unequal recombination events, contribute in comparable proportions to duplication formation, the latter being prone to create novel gene fusions at chromosomal junctions. The mechanisms identified in yeast could explain the origin of a variety of genetic diseases in human, such as hemophilia A, Pelizaeus-Merzbacher disease, or some neurological disorders.