DNA replication errors drive genome-wide small inverted triplication dynamics

Abstract Structural variants (SVs) have a profound impact on phenotype and diversity and are associated with human diseases. To explore the origination of SVs, we have analyzed 1,340 cancer genomes with annotation of 4,608 novel small inverted triplication (SIT) events and found that FEN1 is strongly associated with SIT incidence. Then, we performed long-read sequencing and developed PacBioR to annotate SITs in yeast FEN1 mutant cells. We found that SIT structures mimic classic inverted triplications but with a smaller DUP/IN/DUP structure of 184/160/184 bp on average, with a spacer sequence of 30 bp and breakpoint junction of 6 bp. We further showed that breakpoints of SITs preferentially occurred at nucleosome midpoints, aligned with Okazaki fragment termini, and those harbored in plasmids were precisely eliminated via DNA polymerase slippage over SIT-derived hairpin structures. This study provides mechanistic insight into SIT origination and offers practical tools for future studies on genome rearrangements. Competing Interest Statement The authors have declared no competing interest. Footnotes Funding: This work was supported by funding from the National Cancer Institute (NCI)/National Institutes of Health (NIH) [R01 CA073764 and R01 CA279840 to B.H.S., R50 CA211397 to L.Z.]; and CCSG [P30 CA033572] to City of Hope. Conflict of Interest Disclosure: The authors declare no competing interests.