SUMMARY
Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce ...DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants’ viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non‐replicative polymerase involved in trans‐lesion synthesis during DNA replication and double‐strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants’ responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.
Significance Statement
DNA polymerase theta (Pol θ) is one of the few proteins involved in DNA repair that appears to be essential for plant development, but there are contradictory reports concerning the phenotype of Pol θ‐deficient mutants. Here we show that Pol θ plays a key role in the repair of replication‐associated DNA breaks and that its requirement for plant development depends on growth conditions, providing evidence for a link between abiotic stress responses and the DNA damage response.
Chloroplasts around the plant cell cycle Pedroza-Garcia, José-Antonio; Domenichini, Séverine; Bergounioux, Catherine ...
Current opinion in plant biology,
December 2016, 2016-Dec, 2016-12-00, 20161201, 2016-12, Volume:
34
Journal Article
Peer reviewed
•Synchronization of host and symbiont cell cycle has been a key step for evolution of plastids.•Plastid division is cell cycle-regulated in algae and probably in plants.•Retrograde signalling ...represents a new pathway regulating cell proliferation in plants.
Plastids arose from an endosymbiosis between a host cell and free-living bacteria. One key step during this evolutionary process has been the establishment of coordinated cell and symbiont division to allow the maintenance of organelles during proliferation of the host. However, surprisingly little is known about the underlying mechanisms. In addition, due to their central role in the cell's energetic metabolism and to their sensitivity to various environmental cues such as light or temperature, plastids are ideally fitted to be the source of signals allowing plants to adapt their development according to external conditions. Consistently, there is accumulating evidence that plastid-derived signals can impinge on cell cycle regulation. In this review, we summarize current knowledge of the dialogue between chloroplasts and the nucleus in the context of the cell cycle.
SUMMARY
Being sessile organisms, plants are ubiquitously exposed to stresses that can affect the DNA replication process or cause DNA damage. To cope with these problems, plants utilize DNA damage ...response (DDR) pathways, consisting of both highly conserved and plant‐specific elements. As a part of this DDR, cell cycle checkpoint control mechanisms either pause the cell cycle, to allow DNA repair, or lead cells into differentiation or programmed cell death, to prevent the transmission of DNA errors in the organism through mitosis or to its offspring via meiosis. The two major DDR cell cycle checkpoints control either the replication process or the G2/M transition. The latter is largely overseen by the plant‐specific SOG1 transcription factor, which drives the activity of cyclin‐dependent kinase inhibitors and MYB3R proteins, which are rate limiting for the G2/M transition. By contrast, the replication checkpoint is controlled by different players, including the conserved kinase WEE1 and likely the transcriptional repressor RBR1. These checkpoint mechanisms are called upon during developmental processes, in retrograde signaling pathways, and in response to biotic and abiotic stresses, including metal toxicity, cold, salinity, and phosphate deficiency. Additionally, the recent expansion of research from Arabidopsis to other model plants has revealed species‐specific aspects of the DDR. Overall, it is becoming evidently clear that the DNA damage checkpoint mechanisms represent an important aspect of the adaptation of plants to a changing environment, hence gaining more knowledge about this topic might be helpful to increase the resilience of plants to climate change.
Significance Statement
To cope with stresses that might impede the replication process or cause DNA breaks, plants utilize DNA damage‐responsive cell cycle checkpoint mechanisms that help them adapt to the stress while maintaining genome integrity. Understanding these mechanisms might be pivotal to increase the resilience of plants to climate change.
Plant DNA Polymerases Pedroza-Garcia, Jose-Antonio; De Veylder, Lieven; Raynaud, Cécile
International journal of molecular sciences,
10/2019, Volume:
20, Issue:
19
Journal Article
Peer reviewed
Open access
Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic ...information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10-15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication.
Gene expression is reconfigured rapidly during the cell cycle to execute the cellular functions specific to each phase. Studies conducted with synchronized plant cell suspension cultures have ...identified hundreds of genes with periodic expression patterns across the phases of the cell cycle, but these results may differ from expression occurring in the context of intact organs. Here, we describe the use of fluorescence-activated cell sorting to analyze the gene expression profile of G2/M cells in the growing root. To this end, we isolated cells expressing the early mitosis cell cycle marker
from
root tips. Transcriptome analysis of these cells allowed identification of hundreds of genes whose expression is reduced or enriched in G2/M cells, including many not previously reported from cell suspension cultures. From this dataset, we identified SCL28, a transcription factor belonging to the GRAS family, whose messenger RNA accumulates to the highest levels in G2/M and is regulated by MYB3R transcription factors. Functional analysis indicates that
promotes progression through G2/M and modulates the selection of cell division planes.
Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology ...remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203–424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.
Survival of living organisms is fully dependent on their maintenance of genome integrity, being permanently threatened by replication stress in proliferating cells. Although the plant DNA damage ...response (DDR) regulator SOG1 has been demonstrated to cope with replicative defects, accumulating evidence points to other pathways functioning independently of SOG1. Here, we have studied the role of the Arabidopsis E2FA and EF2B transcription factors, two well-characterized regulators of DNA replication, in the response to replication stress. Through a combination of reverse genetics and chromatin-immunoprecipitation approaches, we show that E2FA and E2FB share many target genes with SOG1, providing evidence for their involvement in the DDR. Analysis of double and triple mutant combinations revealed that E2FB, rather than E2FA, plays the most prominent role in sustaining growth in the presence of replicative defects, either operating antagonistically or synergistically with SOG1. Reversely, SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants. Our data reveal a complex transcriptional network controlling the replication stress response, in which both E2Fs and SOG1 act as key regulatory factors.
Thymidine kinase catalyzes the first step in the nucleotide salvage pathway by transferring a phosphate group to a thymidine molecule. In mammals thymidine kinase supplies deoxyribonucleotides for ...DNA replication and DNA repair, and the expression of the gene is tightly regulated during the cell cycle. Although this gene is phylogenetically conserved in many taxa, its physiological function in plants remains unknown. The genome of the model plant Arabidopsis thaliana has two thymidine kinase genes (AtTK1a and AtTK1b) and microarray data suggest they might have redundant roles. In this study we analyzed the TK1a function by evaluating its expression pattern during development and in response to genotoxic stress. We also studied its role in DNA repair by the characterization of a mutant that contained the T-DNA insertion in the promoter region of the TK1a gene. We found that TK1a is expressed in most tissues during plant development and it was differentially induced by ultraviolet-C radiation because TK1b expression was unaffected. In the mutant, the T-DNA insertion caused a 40 % rise in transcript levels and enzyme activity in Arabidopsis seedlings compared to wild-type plants. This elevation was enough to confer tolerance to ultraviolet-C irradiation in dark conditions, as determined by root growth, and meristem length and structure. TK1a overexpression also provided tolerance to genotoxins that induce double-strand break. Our results suggest that thymidine kinase contributes to several DNA repair pathways by providing deoxythymidine triphosphate that serve as precursors for DNA repair and to balance deoxyribonucleotides pools.
Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of ...replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase 𝜀 (Pol 𝜀) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol 𝜀 and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol 𝜀 trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol 𝜀 in replicative stress sensing. Together, our results demonstrate that the role of Pol 𝜀 in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.
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