Clustering of Inverted Triplications in Centromeric and Subtelomeric Chromosomal Regions of

The formation of inverted repeats frequently initiates rearrangements associated with gene amplification. Among these rearrangements are inverted triplications (TRP/INVs) that are associated with multiple disease states in humans. We have used the filamentous fungus Aspergillus flavus as a model system to analyze spontaneous DNA rearrangements using high-depth third-generation sequencing of DNA isolated from vegetatively cultured strains. Analysis of sequence data identified a class of infrequent and transient rearrangements having structures typical of TRP/INVs. These TRP/INVs form both unprocessed and processed species and have a variably sized deletion at the junction between direct and inverted sequences. We found that TRP/INVs are enriched in heterochromatic centromeric and subtelomeric A+T-rich regions, suggesting that this process is a source of genetic instability in these domains. Consistent with this finding, A+T-rich regions contain elevated levels of direct, inverted, and perfect palindromic repeats. Inverted junctions contain palindromic sequences that are typically associated with TRP/INVs. A closer examination of the class of genomic palindromes found at inverted junctions revealed a broad distribution, with the majority lying in AT-rich centromeric sequences. The distribution of TRP/INVs in centromeric and subtelomeric domains mirrors the frequency of palindromes, indicating that palindrome abundance is likely to be a driver of TRP/INVs. A further examination of predicted pairing patterns in palindrome-like structures suggests that these structures may function as sites of DNA resolution, strand transfer, and replicative stalling. These results are consistent with a replication-based model in which palindromes, under conditions of replication stress, facilitate the formation of TRP/INV structures. These studies also represent, to our knowledge, the first characterization of spontaneous TRP/INV formation, likely to underlie the complex rearrangements associated with higher eukaryotic genomic instability and disease states. AUTHOR SUMMARY: Inverted triplications (TRP/INVs), coupled with gene amplification, are sources of genetic instability. However, little is known about how these species arise spontaneously prior to selective pressures. We have established a fungal model system that uses long-read third-generation sequencing to identify low-abundance rearrangements in the absence of an intentional selection. We identified and characterized multiple classes of TRP/INVs flanked by palindromic sequences. These rearrangements were concentrated in the AT-rich centromeric and subtelomeric regions, suggesting that these sites are regulated recombinational hotspots. Palindromes that are also associated with TRP/INVs are also most prevalent in A+T-rich centromeric regions, suggesting that the abundance of palindromes, both as a source of DNA damage and fork stalling, is a driver of TRP/INV formation. These studies provide the first evidence (to our knowledge) of the spontaneous formation of TRP/INVs during DNA replication, which is likely to precede more complex sources of rearrangements associated with oncogenesis and other disease states in higher eukaryotes.