The repair of topoisomerase 2 cleavage complexes in Arabidopsis

Abstract

DNA–protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2–DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function non-redundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative non-homologous end joining (NHEJ) pathways. Double mutant analysis indicates that “clean” DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes non-ligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.

SMC5/6 complex-mediated SUMOylation stimulates DNA–protein cross-link repair in Arabidopsis

“…Although nse4a-2 seedlings showed a relatively strong reduction in root length (50.3% ± 8.3%), this effect was not significantly different from that of WT seedlings ( P = 0.169), consistent with the classification of the nse4a-2 mutant as a hypomorphic allele ( Diaz et al, 2019 ). In agreement with published data, we observed sensitivity to CPT for mus81-1 and wss1a-1 seedlings, but not for tdp1-3 or tdp2-5 ( Supplemental Figure 5, B and, 5E ) ( Enderle et al, 2019 ; Hacker et al, 2022 ).…”

“…In all cases with significantly reduced root length, we also detected more dead cells in the root meristems of smc6b-1 , multiple alleles of hze1 and nse4a-2 seedlings ( Figure 2D ). We also observed hypersensitivity to ICRF-187 in wss1a-1, which was in agreement with the known function of WSS1A in the repair of TOP2 cross-links ( Hacker et al, 2022 ) ( Supplemental Figure 5, C–E , Supplemental Table 3 ). ICRF-187 is a highly specific TOP2 inhibitor that links the interface between two ATPase protomers of TOP2 ( Classen et al, 2003 ; Nitiss, 2009 ; Lee et al, 2017 , 2022 ).…”

“…This type of DPC is also caused by the TOP2 poison etoposide, which inhibits the religation of cleaved DNA segments, resulting in TOP2 binding to the cleaved DNA ends (reviewed in Nitiss, 2009 ). Because etoposide shortens root length only at very high concentrations ( Hacker et al, 2022 ), we screened several other TOP2 poisons and inhibitors used in mammalian research and observed a strong effect after treatment with bisdioxopiperazine dexrazoxane (ICRF-187) ( Figure 2, B–D , Supplemental Table 2 ). Seedlings from smc6b-1 , nse4a-2, and all tested hze1 alleles (− 1 to − 3 ) showed a massive root length reduction to less than 25% of mock-treated controls, while the root length in WT seedlings was only weakly affected in response to 10 µM ICRF-187, with a root length of 81.0% ± 1.7% relative to the mock treatment.…”

“…In contrast to animals, TDP1 only plays a minor role in the repair of TOP1 cross-links in Arabidopsis and may function as a backup pathway to MUS81 and WSS1A-mediated repair ( Enderle et al, 2019 ). Additionally, TDP2 contributes to the repair of TOP2 cross-links in Arabidopsis ( Hacker et al, 2022 ). Last, DPCs can be directly processed by DNA endonucleases.…”

The Role of Topoisomerase II in DNA Repair and Recombination in Arabidopsis thaliana

“…In both prokaryotes and eukaryotes, type II topoisomerases consist of a homodimer that requires ATP and Mg +2 to perform its catalytic activity: they bind to a region of the DNA called the G-segment and trap a second region of DNA, the T segment; ATP hydrolysis helps G-segment cleavage (DNA double-strand break formation), letting the T segment pass in between the broken ends. The DNA-protein crosslink created by the activity of TOPII, normally repaired by Tyrosyl-DNA-phosphodiesterase 2 [ 10 ], can cause DSBs. After releasing the DNA, the homodimer then returns to an open position.…”