Breadth by depth: Expanding our understanding of the repair of transposon-induced DNA double strand breaks via deep-sequencing

• Published in: DNA repair 2011-10, Vol.10 (10), p.1023-1033
• Main Authors: Huefner, Neil D., Mizuno, Yurie, Weil, Clifford F., Korf, Ian, Britt, Anne B.
• Format: Article
• Language: English
• Subjects: Ac element excision; alt-NHEJ; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Bacteriology; Base Sequence; Biological and medical sciences; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases - genetics; DNA Ligases - metabolism; DNA Transposable Elements - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Genic rearrangement. Recombination. Transposable element; Growth, nutrition, cell differenciation; Hairpin end; High-Throughput Nucleotide Sequencing; Inverted Repeat Sequences; Microbiology; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis. Repair; Sequence Analysis, DNA; Zea mays - genetics; Zea mays - metabolism
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2011.07.011

[Display omitted] ► Excision of the transposable element Ac creates a hairpin-ended DSB. ► We employed Illumina sequencing to study the effects of repair and response defects on the mutations generated by Ac excision. ► 261,000 excision products were analyzed. In wild-type a single product constitutes half of all repair events. ► Defects in NHEJ factors ( ku80, lig4), polymerases ( polL) and a Pso2/ARTEMIS homologue ( lig6) reduced the accuracy of repair. ► Knockouts in ATM or ATR had similar effects on mutation spectrum, suggesting that both kinases are routinely involved in repair. The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed.