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 nonredundantly 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 nonhomologous 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 nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , DNA Topoisomerases/genetics , Arabidopsis/enzymology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , DNA Topoisomerases/metabolismABSTRACT
The protease WSS1A is an important factor in the repair of DNA-protein crosslinks in plants. Here we show that the loss of WSS1A leads to a reduction of 45S rDNA repeats and chromosomal fragmentation in Arabidopsis. Moreover, in the absence of any factor of the RTR (RECQ4A/TOP3α/RMI1/2) complex, which is involved in the dissolution of DNA replication intermediates, WSS1A becomes essential for viability. If WSS1A loss is combined with loss of the classical (c) or alternative (a) nonhomologous end joining (NHEJ) pathways of double-strand break (DSB) repair, the resulting mutants show proliferation defects and enhanced chromosome fragmentation, which is especially aggravated in the absence of aNHEJ. This indicates that WSS1A is involved either in the suppression of DSB formation or in DSB repair itself. To test the latter we induced DSB by CRISPR/Cas9 at different loci in wild-type and mutant cells and analyzed their repair by deep sequencing. However, no change in the quality of the repair events and only a slight increase in their quantity 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 DSB leading to genome instability.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , DNA , DNA End-Joining Repair , DNA Repair , Peptide Hydrolases/metabolismABSTRACT
DNA-protein crosslinks represent a severe kind of DNA damage as they disturb essential processes, such as transcription and DNA replication, due to their bulkiness. To ensure the maintenance of genome integrity, it is necessary for all living organisms to repair these lesions in a timely manner. Over recent years, much knowledge has been obtained regarding the repair of DNA-protein crosslinks (DPC), but it was only recently that the first insights into the mechanisms of DPC repair in plants were obtained. The plant DPC repair network consists of at least three parallel pathways that resolve DPC by distinct biochemical mechanisms. The endonuclease MUS81 resolves the DPC by cleaving the DNA part of the crosslink, the protease WSS1A is able to degrade the protein part and the tyrosyl-DNA-phosphodiesterase TDP1 can hydrolyse the crosslink between a protein and the DNA. However, due to the variety of different DPC types and the evolutionary conservation of pathways between eukaryotes, we expect that future research will reveal additional factors involved in DPC repair in plants.