Clustering of Inverted Triplications in Centromeric and Subtelomeric Chromosomal Regions of Aspergillus flavus

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.

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.