RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , ADN-Topoisomerasas/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , ADN-Topoisomerasas/metabolismoRESUMEN
The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by â¼52% in msh5/zyp1 compared to msh5 These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Intercambio Genético , Arabidopsis , Proteínas de Arabidopsis/genética , Meiosis , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismoRESUMEN
DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologs of the universal repair proteases SPARTAN/ weak suppressor of smt3 (Wss1), WSS1A, is essential for DPC repair in Arabidopsis (Arabidopsis thaliana). WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of TYROSYL-DNA PHOSPHODIESTERASE 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Mutación/genéticaRESUMEN
Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases.
Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN/fisiología , Replicación del ADN , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Anemia de Fanconi/genética , Inestabilidad Genómica , Meristema/metabolismo , Mutación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , ARN Helicasas/genéticaRESUMEN
DNA-crosslinks are one of the most severe types of DNA lesions. Crosslinks (CLs) can be subdivided into DNA-intrastrand CLs, DNA-interstrand CLs (ICLs) and DNA-protein crosslinks (DPCs), and arise by various exogenous and endogenous sources. If left unrepaired before the cell enters S-phase, ICLs and DPCs pose a major threat to genomic integrity by blocking replication. In order to prevent the collapse of replication forks and impairment of cell division, complex repair pathways have emerged. In mammals, ICLs are repaired by the so-called Fanconi anemia (FA) pathway, which includes 22 different FANC genes, while in plants only a few of these genes are conserved. In this context, two pathways of ICL repair have been defined, each requiring the interaction of a helicase (FANCJB/RTEL1) and a nuclease (FAN1/MUS81). Moreover, homologous recombination (HR) as well as postreplicative repair factors are also involved. Although DPCs possess a comparable toxic potential to cells, it has only recently been shown that at least three parallel pathways for DPC repair exist in plants, defined by the protease WSS1A, the endonuclease MUS81 and tyrosyl-DNA phosphodiesterase 1 (TDP1). The importance of crosslink repair processes are highlighted by the fact that deficiencies in the respective pathways are associated with diverse hereditary disorders.
Asunto(s)
ADN/metabolismo , Anemia de Fanconi/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Anemia de Fanconi/genética , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismoRESUMEN
RecQ helicases are important caretakers of genome stability and occur in varying copy numbers in different eukaryotes. Subsets of RecQ paralogs are involved in DNA crosslink (CL) repair. The orthologs of AtRECQ2, AtRECQ3 and AtHRQ1, HsWRN, DmRECQ5 and ScHRQ1 participate in CL repair in their respective organisms, and we aimed to define the function of these helicases for plants. We obtained Arabidopsis mutants of the three RecQ helicases and determined their sensitivity against CL agents in single- and double-mutant analyses. Only Athrq1, but not Atrecq2 and Atrecq3, mutants proved to be sensitive to intra- and interstrand crosslinking agents. AtHRQ1 is specifically involved in the repair of replicative damage induced by CL agents. It shares pathways with the Fanconi anemia-related endonuclease FAN1 but not with the endonuclease MUS81. Most surprisingly, AtHRQ1 is epistatic to the ATPase RAD5A for intra- as well as interstrand CL repair. We conclude that, as in fungi, AtHRQ1 has a conserved function in DNA excision repair. Additionally, HRQ1 not only shares pathways with the Fanconi anemia repair factors, but in contrast to fungi also seems to act in a common pathway with postreplicative DNA repair.