Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA

by Rebecca Martin, Claudia Y. Espinoza, Christopher R. L. Large, Joshua Rosswork, Cole Van Bruinisse, Aaron W. Miller, Joseph C. Sanchez, Madison Miller, Samantha Paskvan, Gina M. Alvino, Maitreya J. Dunham, M. K. Raghuraman, Bonita J. Brewer Inherited and germ-linede novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA —origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split -ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres —suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initia ting event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyure...
Source: PLoS Genetics - Category: Genetics & Stem Cells Authors: Source Type: research