The role of DNA-protein crosslink repair in meiotic recombination, T-DNA integration and gene editing
Final Report Abstract
Although DNA-protein crosslinks (DPCs) represent a severe threat to genome integrity, it was only recently that the main mechanisms of DPC repair were elucidated in humans and yeast. By the use of CRISPR/Cas9 mutants, we able to define already before the start of the project basic features of DPC repair in plants and could show that a homologue of the universal repair proteases SPRTN/WSS1 (WSS1A) is essential for this process in Arabidopsis. In the current project we were able to elucidate the important role of DPC repair on plant genome stability in detail. On one side we were able to define how topoisomerase 2 DPC intermediates (TOP2cc) are repaired in plants. They are a very serious threat to genome stability as double strand breaks (DSBs) arise as repair intermediates. Our analysis shows that there exist two main pathways of TOP2cc repair in Arabidopsis: One is defined by the Tyrosyl-DNA-Phosphodiesterase 2 (AtTDP2) which is able to hydrolyze TOP2-DNA linkages, the other by WSS1A. In both pathways DSBs arise, which are subsequently repaired by both known pathways of non-homologous end joining (NHEJ). Double mutant analysis indicates that “clean” DNA ends, caused by AtTDP2 hydrolysis, are mainly religated by classical (c)NHEJ which promotes mutation avoidance. In contrast, the mutagenic alternative (a)NHEJ pathway is mainly involved in processing DNA ends that are not directly ligateable. Furthermore, we characterized the role of WSS1A on other aspects of genome stability in detail. In absence of any factor of the RTR complex, which is involved in the dissolution of DNA replication intermediates, WSS1A becomes essential for viability. The severe reduction of 45S rDNA repeats is also an indication of defects during DNA replication. Loss of the protease leads to a chromosomal fragmentation in somatic cells of Arabidopsis. Due to this somatic defect, cells with unrepaired fragmented chromosomes enter meiosis, resulting in a reduction of fertility. If WSS1A loss is combined with loss of cNHEJ or aNHEJ pathways of DSB repair, the resulting mutants show proliferation defects and enhanced chromosome fragmentation, which is especially aggravated in absence of aNHEJ. This indicates that WSS1A is either involved in the suppression of DSB formation or in DSB repair itself. To test the latter, we induced DSBs by CRISPR/Cas9 at different loci in WT and mutant cells and analyzed their repair by deep sequencing. However, no mayor change in the quality or quantity of the repair events was found. Thus, by removing complex DNA-protein structures, WSS1A seems to be required for the repair of replication intermediates which would otherwise be resolved into persistent DSBs leading to genome instability.
Publications
-
Repair of DNA-protein crosslinks in plants. DNA Repair, 87, 102787.
Hacker, Leonie; Dorn, Annika & Puchta, Holger
-
The DNA‐dependent protease AtWSS1A suppresses persistent double strand break formation during replication. New Phytologist, 233(3), 1172-1187.
Hacker, Leonie; Capdeville, Niklas; Feller, Laura; Enderle‐Kukla, Janina; Dorn, Annika & Puchta, Holger
-
The repair of topoisomerase 2 cleavage complexes in Arabidopsis. The Plant Cell, 34(1), 287-301.
Hacker, Leonie; Dorn, Annika; Enderle, Janina & Puchta, Holger
