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13 pages, 1855 KiB

Open AccessArticle

Quantification of 8-oxoG in Plant Telomeres

by Claudia Castillo-González, Borja Barbero Barcenilla, Pierce G. Young, Emily Hall and Dorothy E. Shippen

Int. J. Mol. Sci. 2022, 23(9), 4990; https://doi.org/10.3390/ijms23094990 - 30 Apr 2022

Cited by 6 | Viewed by 2481

Abstract

Chemical modifications in DNA impact gene regulation and chromatin structure. DNA oxidation, for example, alters gene expression, DNA synthesis and cell cycle progression. Modification of telomeric DNA by oxidation is emerging as a marker of genotoxic damage and is associated with reduced genome [...] Read more.

Chemical modifications in DNA impact gene regulation and chromatin structure. DNA oxidation, for example, alters gene expression, DNA synthesis and cell cycle progression. Modification of telomeric DNA by oxidation is emerging as a marker of genotoxic damage and is associated with reduced genome integrity and changes in telomere length and telomerase activity. 8-oxoguanine (8-oxoG) is the most studied and common outcome of oxidative damage in DNA. The G-rich nature of telomeric DNA is proposed to make it a hotspot for oxidation, but because telomeres make up only a tiny fraction of the genome, it has been difficult to directly test this hypothesis by studying dynamic DNA modifications specific to this region in vivo. Here, we present a new, robust method to differentially enrich telomeric DNA in solution, coupled with downstream methods for determination of chemical modification. Specifically, we measure 8-oxoG in Arabidopsis thaliana telomeres under normal and oxidative stress conditions. We show that telomere length is unchanged in response to oxidative stress in three different wild-type accessions. Furthermore, we report that while telomeric DNA comprises only 0.02–0.07% of the total genome, telomeres contribute between 0.2 and 15% of the total 8-oxoG. That is, plant telomeres accumulate 8-oxoG at levels approximately 100-fold higher than the rest of the genome under standard growth conditions. Moreover, they are the primary targets of further damage upon oxidative stress. Interestingly, the accumulation of 8-oxoG in the chromosome body seems to be inversely proportional to telomere length. These findings support the hypothesis that telomeres are hotspots of 8-oxoG and may function as sentinels of oxidative stress in plants. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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13 pages, 1831 KiB

Open AccessArticle

The Barley Chloroplast Mutator (cpm) Mutant: All Roads Lead to the Msh1 Gene

by Franco Lencina, Alejandra Landau and Alberto R. Prina

Int. J. Mol. Sci. 2022, 23(3), 1814; https://doi.org/10.3390/ijms23031814 - 05 Feb 2022

Cited by 3 | Viewed by 1970

Abstract

The barley chloroplast mutator (cpm) is a nuclear gene mutant that induces a wide spectrum of cytoplasmically inherited chlorophyll deficiencies. Plastome instability of cpm seedlings was determined by identification of a particular landscape of polymorphisms that suggests failures in a plastome [...] Read more.

The barley chloroplast mutator (cpm) is a nuclear gene mutant that induces a wide spectrum of cytoplasmically inherited chlorophyll deficiencies. Plastome instability of cpm seedlings was determined by identification of a particular landscape of polymorphisms that suggests failures in a plastome mismatch repair (MMR) protein. In Arabidopsis, MSH genes encode proteins that are in charge of mismatch repair and have anti-recombination activity. In this work, barley homologs of these genes were identified, and their sequences were analyzed in control and cpm mutant seedlings. A substitution, leading to a premature stop codon and a truncated MSH1 protein, was identified in the Msh1 gene of cpm plants. The relationship between this mutation and the presence of chlorophyll deficiencies was established in progenies from crosses and backcrosses. These results strongly suggest that the mutation identified in the Msh1 gene of the cpm mutant is responsible for the observed plastome instabilities. Interestingly, comparison of mutant phenotypes and molecular changes induced by the barley cpm mutant with those of Arabidopsis MSH1 mutants revealed marked differences. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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16 pages, 3237 KiB

Open AccessArticle

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

by Marina Martinez-Garcia, Charles I. White, F. Chris. H. Franklin and Eugenio Sanchez-Moran

Int. J. Mol. Sci. 2021, 22(23), 13115; https://doi.org/10.3390/ijms222313115 - 04 Dec 2021

Cited by 3 | Viewed by 3077

Abstract

DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, [...] Read more.

DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of Arabidopsis thaliana TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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15 pages, 1878 KiB

Open AccessArticle

Targeted Inter-Homologs Recombination in Arabidopsis Euchromatin and Heterochromatin

by Shdema Filler-Hayut, Kiril Kniazev, Cathy Melamed-Bessudo and Avraham A. Levy

Int. J. Mol. Sci. 2021, 22(22), 12096; https://doi.org/10.3390/ijms222212096 - 09 Nov 2021

Cited by 7 | Viewed by 2120

Abstract

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA [...] Read more.

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both “hot and cold” regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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10 pages, 1603 KiB

Open AccessArticle

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing

by Paul A. P. Atkins, Maria Elena S. Gamo and Daniel F. Voytas

Int. J. Mol. Sci. 2021, 22(18), 9723; https://doi.org/10.3390/ijms22189723 - 08 Sep 2021

Cited by 1 | Viewed by 2719

Abstract

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such [...] Read more.

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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12 pages, 3028 KiB

Open AccessArticle

A Mutation in DNA Polymerase α Rescues WEE1^KO Sensitivity to HU

by Thomas Eekhout, José Antonio Pedroza-Garcia, Pooneh Kalhorzadeh, Geert De Jaeger and Lieven De Veylder

Int. J. Mol. Sci. 2021, 22(17), 9409; https://doi.org/10.3390/ijms22179409 - 30 Aug 2021

Cited by 3 | Viewed by 1926

Abstract

During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication [...] Read more.

During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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23 pages, 6871 KiB

Open AccessArticle

Heterologous Complementation of SPO11-1 and -2 Depends on the Splicing Pattern

by Thorben Sprink and Frank Hartung

Int. J. Mol. Sci. 2021, 22(17), 9346; https://doi.org/10.3390/ijms22179346 - 28 Aug 2021

Cited by 3 | Viewed by 2317

Abstract

In the past, major findings in meiosis have been achieved, but questions towards the global understanding of meiosis remain concealed. In plants, one of these questions covers the need for two diverse meiotic active SPO11 proteins. In Arabidopsis and other plants, both meiotic [...] Read more.

In the past, major findings in meiosis have been achieved, but questions towards the global understanding of meiosis remain concealed. In plants, one of these questions covers the need for two diverse meiotic active SPO11 proteins. In Arabidopsis and other plants, both meiotic SPO11 are indispensable in a functional form for double strand break induction during meiotic prophase I. This stands in contrast to mammals and fungi, where a single SPO11 is present and sufficient. We aimed to investigate the specific function and evolution of both meiotic SPO11 paralogs in land plants. By performing immunostaining of both SPO11-1 and -2, an investigation of the spatiotemporal localization of each SPO11 during meiosis was achieved. We further exchanged SPO11-1 and -2 in Arabidopsis and could show a species-specific function of the respective SPO11. By additional changes of regions between SPO11-1 and -2, a sequence-specific function for both the SPO11 proteins was revealed. Furthermore, the previous findings about the aberrant splicing of each SPO11 were refined by narrowing them down to a specific developmental phase. These findings let us suggest that the function of both SPO11 paralogs is highly sequence specific and that the orthologs are species specific. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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15 pages, 3796 KiB

Open AccessArticle

Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs

by Marina Jordano-Raya, Cristina Beltrán-Melero, M. Dolores Moreno-Recio, M. Isabel Martínez-Macías, Rafael R. Ariza, Teresa Roldán-Arjona and Dolores Córdoba-Cañero

Int. J. Mol. Sci. 2021, 22(16), 8763; https://doi.org/10.3390/ijms22168763 - 16 Aug 2021

Cited by 2 | Viewed by 1864

Abstract

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. [...] Read more.

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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Review

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12 pages, 868 KiB

Open AccessReview

Multiple Roles of SMC5/6 Complex during Plant Sexual Reproduction

by Fen Yang and Ales Pecinka

Int. J. Mol. Sci. 2022, 23(9), 4503; https://doi.org/10.3390/ijms23094503 - 19 Apr 2022

Cited by 2 | Viewed by 2103

Abstract

Chromatin-based processes are essential for cellular functions. Structural maintenance of chromosomes (SMCs) are evolutionarily conserved molecular machines that organize chromosomes throughout the cell cycle, mediate chromosome compaction, promote DNA repair, or control sister chromatid attachment. The SMC5/6 complex is known for its pivotal [...] Read more.

Chromatin-based processes are essential for cellular functions. Structural maintenance of chromosomes (SMCs) are evolutionarily conserved molecular machines that organize chromosomes throughout the cell cycle, mediate chromosome compaction, promote DNA repair, or control sister chromatid attachment. The SMC5/6 complex is known for its pivotal role during the maintenance of genome stability. However, a dozen recent plant studies expanded the repertoire of SMC5/6 complex functions to the entire plant sexual reproductive phase. The SMC5/6 complex is essential in meiosis, where its activity must be precisely regulated to allow for normal meiocyte development. Initially, it is attenuated by the recombinase RAD51 to allow for efficient strand invasion by the meiosis-specific recombinase DMC1. At later stages, it is essential for the normal ratio of interfering and non-interfering crossovers, detoxifying aberrant joint molecules, preventing chromosome fragmentation, and ensuring normal chromosome/sister chromatid segregation. The latter meiotic defects lead to the production of diploid male gametes in Arabidopsis SMC5/6 complex mutants, increased seed abortion, and production of triploid offspring. The SMC5/6 complex is directly involved in controlling normal embryo and endosperm cell divisions, and pioneer studies show that the SMC5/6 complex is also important for seed development and normal plant growth in cereals. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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15 pages, 1679 KiB

Open AccessReview

Genome Maintenance Mechanisms at the Chromatin Level

by Hirotomo Takatsuka, Atsushi Shibata and Masaaki Umeda

Int. J. Mol. Sci. 2021, 22(19), 10384; https://doi.org/10.3390/ijms221910384 - 27 Sep 2021

Cited by 3 | Viewed by 2800

Abstract

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling [...] Read more.

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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16 pages, 486 KiB

Open AccessReview

Connections between the Cell Cycle and the DNA Damage Response in Plants

by Naomie Gentric, Pascal Genschik and Sandra Noir

Int. J. Mol. Sci. 2021, 22(17), 9558; https://doi.org/10.3390/ijms22179558 - 03 Sep 2021

Cited by 14 | Viewed by 5304

Abstract

Due to their sessile lifestyle, plants are especially exposed to various stresses, including genotoxic stress, which results in altered genome integrity. Upon the detection of DNA damage, distinct cellular responses lead to cell cycle arrest and the induction of DNA repair mechanisms. Interestingly, [...] Read more.

Due to their sessile lifestyle, plants are especially exposed to various stresses, including genotoxic stress, which results in altered genome integrity. Upon the detection of DNA damage, distinct cellular responses lead to cell cycle arrest and the induction of DNA repair mechanisms. Interestingly, it has been shown that some cell cycle regulators are not only required for meristem activity and plant development but are also key to cope with the occurrence of DNA lesions. In this review, we first summarize some important regulatory steps of the plant cell cycle and present a brief overview of the DNA damage response (DDR) mechanisms. Then, the role played by some cell cycle regulators at the interface between the cell cycle and DNA damage responses is discussed more specifically. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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16 pages, 1586 KiB

Open AccessReview

Plant DNA Repair and Agrobacterium T−DNA Integration

by Stanton B. Gelvin

Int. J. Mol. Sci. 2021, 22(16), 8458; https://doi.org/10.3390/ijms22168458 - 06 Aug 2021

Cited by 23 | Viewed by 6424

Abstract

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, [...] Read more.

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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11 pages, 2484 KiB

Open AccessReview

Boon and Bane of DNA Double-Strand Breaks

by Ingo Schubert

Int. J. Mol. Sci. 2021, 22(10), 5171; https://doi.org/10.3390/ijms22105171 - 13 May 2021

Cited by 11 | Viewed by 3080

Abstract

DNA double-strand breaks (DSBs), interrupting the genetic information, are elicited by various environmental and endogenous factors. They bear the risk of cell lethality and, if mis-repaired, of deleterious mutation. This negative impact is contrasted by several evolutionary achievements for DSB processing that help [...] Read more.

DNA double-strand breaks (DSBs), interrupting the genetic information, are elicited by various environmental and endogenous factors. They bear the risk of cell lethality and, if mis-repaired, of deleterious mutation. This negative impact is contrasted by several evolutionary achievements for DSB processing that help maintaining stable inheritance (correct repair, meiotic cross-over) and even drive adaptation (immunoglobulin gene recombination), differentiation (chromatin elimination) and speciation by creating new genetic diversity via DSB mis-repair. Targeted DSBs play a role in genome editing for research, breeding and therapy purposes. Here, I survey possible causes, biological effects and evolutionary consequences of DSBs, mainly for students and outsiders. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Plants 2.0)

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