RESUMEN
DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Sumoilación , Reparación del ADN/genética , Daño del ADN , Proteínas/metabolismo , ADN/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Unión al ADN/genética , Reparación del ADN/genética , Marcación de Gen , Reparación del ADN por Unión de ExtremidadesRESUMEN
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 RTR (RecQ/Top3/Rmi1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyper-recombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: the top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Reparación del ADN/genética , Meiosis/genética , Proteínas de Plantas/metabolismo , Solanum lycopersicum/genética , Arabidopsis/enzimología , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Inestabilidad Genómica , Recombinación Homóloga , Solanum lycopersicum/enzimología , Solanum lycopersicum/fisiología , Mutación , Fenotipo , Proteínas de Plantas/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Péptido Hidrolasas/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
Topoisomerase 3α, a class I topoisomerase, consists of a TOPRIM domain, an active centre and a variable number of zinc-finger domains (ZFDs) at the C-terminus, in multicellular organisms. Whereas the functions of the TOPRIM domain and the active centre are known, the specific role of the ZFDs is still obscure. In contrast to mammals where a knockout of TOP3α leads to lethality, we found that CRISPR/Cas induced mutants in Arabidopsis are viable but show growth retardation and meiotic defects, which can be reversed by the expression of the complete protein. However, complementation with AtTOP3α missing either the TOPRIM-domain or carrying a mutation of the catalytic tyrosine of the active centre leads to embryo lethality. Surprisingly, this phenotype can be overcome by the simultaneous removal of the ZFDs from the protein. In combination with a mutation of the nuclease AtMUS81, the TOP3α knockout proved to be also embryo lethal. Here, expression of TOP3α without ZFDs, and in particular without the conserved ZFD T1, leads to only a partly complementation in root growth-in contrast to the complete protein, that restores root length to mus81-1 mutant level. Expressing the E. coli resolvase RusA in this background, which is able to process Holliday junction (HJ)-like recombination intermediates, we could rescue this root growth defect. Considering all these results, we conclude that the ZFD T1 is specifically required for targeting the topoisomerase activity to HJ like recombination intermediates to enable their processing. In the case of an inactivated enzyme, this leads to cell death due to the masking of these intermediates, hindering their resolution by MUS81.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Reparación del ADN/fisiología , ADN-Topoisomerasas de Tipo I/metabolismo , Endonucleasas/metabolismo , Dedos de Zinc/genética , Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Dominio Catalítico/genética , ADN-Topoisomerasas de Tipo I/genética , ADN Cruciforme/genética , Endonucleasas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Resolvasas de Unión Holliday/genética , Resolvasas de Unión Holliday/metabolismo , Mutagénesis , Fenotipo , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas GenéticamenteRESUMEN
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
Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error-free branch of post-replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication-associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single-strand break repair (AtPARP1), as well as microhomology-mediated double-strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM-mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Reparación del ADN , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Dominios Proteicos , UbiquitinaciónRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Reactivos de Enlaces Cruzados/química , ADN Helicasas/genética , Reparación del ADN , Replicación del ADN , Exodesoxirribonucleasas/metabolismo , Anemia de Fanconi/enzimología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Muerte Celular/efectos de los fármacos , Cisplatino/farmacología , ADN Helicasas/metabolismo , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Endodesoxirribonucleasas , Epistasis Genética/efectos de los fármacos , Genoma de Planta , Meristema/efectos de los fármacos , Meristema/fisiología , Mitomicina/farmacología , Enzimas Multifuncionales , Mutación/genética , RecQ Helicasas/metabolismoRESUMEN
CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.
Asunto(s)
Arabidopsis , Sistemas CRISPR-Cas , Arabidopsis/genética , Arabidopsis/metabolismo , Sistemas CRISPR-Cas/genética , Endonucleasas/metabolismo , Mutagénesis , Secuencias Repetidas en TándemRESUMEN
Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR-Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR-Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant chromosome, but also of novel plant species.
Asunto(s)
Sistemas CRISPR-Cas , Cromosomas de las Plantas/genética , Producción de Cultivos , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Biología Sintética , Producción de Cultivos/métodos , Productos Agrícolas/genética , Biología Sintética/métodosRESUMEN
Meiotic and somatic recombination share a common set of factors. Thus, the analysis of somatic DNA repair in meiotic mutant lines should be of special interest. Growth defects of mutant plants induced by specific genotoxins can thereby hint to DNA repair functions of the affected proteins. Here, we describe two kinds of approaches to characterize deficiencies in DNA repair in mutant lines of Arabidopsis thaliana, after genotoxin treatment.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Reparación del ADN , Meiosis/genética , Mutación , Muerte Celular/genética , Meristema/genética , Raíces de Plantas/genéticaRESUMEN
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.
Asunto(s)
Daño del ADN , Reparación del ADN , Plantas/genética , ADN-Topoisomerasas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Endonucleasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
CRISPR/Cas is in the process of inducing the biggest transformation of plant breeding since the green revolution. Whereas initial efforts focused mainly on changing single traits by error prone non-homologous end joining, the last two years saw a tremendous technical progress achieving more complex genetic, epigenetic and transcriptional changes. The efficiencies of inducing directed changes by homologous recombination have been improved significantly and strategies to break genetic linkages by inducing chromosomal rearrangements have been developed. Cas13 systems have been applied to degrade viral and mRNA in plants. Most importantly, a historical breakthrough was accomplished: By introducing multiple genomic changes simultaneously, domestication of wild species in a single generation has been demonstrated, speeding up breeding dramatically.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cruzamiento , Edición Génica , Recombinación Homóloga , Fitomejoramiento , Plantas/genéticaRESUMEN
Genetic information of all organisms is coded in double-stranded DNA. DNA helicases are essential for unwinding this double strand when it comes to replication, repair or transcription of genetic information. In this review, we will focus on what is known about a variety of DNA helicases that are required to ensure genome stability in plants. Due to their sessile lifestyle, plants are especially exposed to harmful environmental factors. Moreover, many crop plants have large and highly repetitive genomes, making them absolutely dependent on the correct interplay of DNA helicases for safeguarding their stability. Although basic features of a number of these enzymes are conserved between plants and other eukaryotes, a more detailed analysis shows surprising peculiarities, partly also between different plant species. This is additionally of high relevance for plant breeding as a number of these helicases are also involved in crossover control during meiosis and influence the outcome of different approaches of CRISPR/Cas based plant genome engineering. Thus, gaining knowledge about plant helicases, their interplay, as well as the manipulation of their pathways, possesses the potential for improving agriculture. In the long run, this might even help us cope with the increasing obstacles of climate change threatening food security in completely new ways.
Asunto(s)
ADN Helicasas/metabolismo , Genoma de Planta/genética , Inestabilidad Genómica/fisiología , ADN/metabolismo , ADN Helicasas/genética , Reparación del ADN/fisiología , Genoma de Planta/fisiología , Inestabilidad Genómica/genética , Meiosis/genética , Plantas/enzimología , RecQ Helicasas/metabolismo , RecQ Helicasas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Two putative adhesion genes of the potentially probiotic strain Lactobacillus plantarum BFE 5092, i.e., a gene with similarity to an aggregation-promoting factor gene apf5092, and the mucin-binding protein gene mub5092, were investigated in this study. The gene encoding apf5092 encoded a protein bearing a predicted 26 amino acid signal peptide and a LysM domain putatively involved in binding to peptidoglycan. Moreover, the deduced protein also showed an amino acid sequence characteristic of an aggregation-promoting factor. The genes were tested for expression under different environmental conditions, and transcriptional studies on apf5092 showed that the expression could be influenced by low temperature and pH within 30 min. The aggregation behavior of the cells also changed at the low pH condition, but less noticeably at low temperature. To further investigate the role of apf5092 in aggregation, it was cloned and expressed in E. coli. The transformed strain showed higher co-aggregation ability with Gram-positive bacteria. Transcription studies on mub5092 revealed that it could be induced by mucin when added to the growth medium within 30 min. The data suggested that L. plantarum BFE 5092 can quickly adapt to changing environmental conditions and that enhanced aggregation may be important to survive low pH conditions, e.g., of the stomach or of fermented foods, and may thus be an important colonization factor.