Biology subjects

Da Ines, O.

Publications and source records attributed to Da Ines, O..

6 recordsLinked to original sources

bioRxiv2026

Role of small non-coding RNAs in DSB repair in Arabidopsis thaliana

DNA double-strand breaks (DSB) are highly toxic lesions that must be repaired to avoid major losses of genetic materials or genome rearrangements. A number of highly conserved molecular pathways are involved in DSB repair, each resulting in different repair outcomes for a given DSB. Over the past decade, accumulating evidence has revealed the emergence of an unexpected new actor in DSB repair: RNA. Notably, small RNAs have been observed at DSB-flanking sequences and hypothesized to be involved in their repair. However, these DSB-induced small RNAs (diRNAs) were so far observed in studies using reporter systems, and their natural occurrence as well as their role in DNA repair remains debated. To address this question, we investigated diRNA biogenesis in Arabidopsis thaliana using CRISPR/Cas12a to induce targeted DSBs at three actively transcribed loci and one untranscribed locus. We found that 21-nt diRNAs are generated at transcribed loci, in a Dicer-like 4 (DCL4) dependent manner, but not at the untranscribed locus, indicating that active transcription is required for diRNA production. Remarkably, long-read DNA sequencing analysis shows that these diRNAs do not detectably influence DSB repair via either end-joining or microhomology-mediated end-joining. Furthermore, endogenous diRNAs have a modest effect on homologous recombination-associated repair, as impairment of the diRNA pathway in ago2 mutants resulted in only a slight reduction in RAD51 focus formation following Cas12a-induced DSBs. Together, these findings indicate that diRNAs are not essential for DSB repair itself but may function in the post-transcriptional silencing of damaged genes, likely to prevent aberrant transcription until proper repair is completed.

genetics↗

bioRxiv2025

Dual role of Arabidopsis SRS2 helicase in meiotic recombination

Helicases are enzymes that use the energy derived from ATP hydrolysis to translocate along and unwind nucleic acids. Accordingly, helicases are instrumental in maintaining genomic integrity and ensuring genetic diversity. Srs2 is a multi-functional DNA helicase that dismantles Rad51 nucleofilaments and regulates DNA strand invasion to prevent excessive or inappropriate homologous recombination in yeast. Consistently, the deletion of Srs2 has significant consequences for the maintenance of genome integrity in mitotic cells. In contrast, its role in meiotic recombination remains less clear. We present here substantial evidence that SRS2 plays an important role in meiotic recombination in the model plant Arabidopsis thaliana. Arabidopsis srs2 mutants exhibit moderate defects in DNA damage-induced RAD51 focus formation, but SRS2 is dispensable for DNA repair and RAD51-dependent recombination in somatic cells. Meiotic progression and fertility appear unaffected in srs2 plants but strikingly, the absence of SRS2 leads to increased genetic interference accompanied by increased numbers of Class I COs and a reduction in MUS81-dependent Class II COs. SRS2 thus has both anti- and pro-CO roles during meiosis in Arabidopsis thaliana - acting as an anti-CO factor by influencing the stability and/or the dissociation of early recombination intermediates, while playing a pro-CO role by promoting MUS81-mediated resolution of meiotic recombination intermediates. Author SummaryHelicases are enzymes that use ATP to unwind DNA. They play a crucial role in maintaining genomic stability. One such helicase, Srs2, is known to regulate homologous recombination in yeast by preventing excessive or inappropriate recombination events. While its function in mitotic cell division is well understood, its role in meiosis - the process that generates reproductive cells - remains less clear. We show here that SRS2 plays a significant role in meiotic recombination in the plant Arabidopsis thaliana. Although Arabidopsis plants lacking SRS2 do not exhibit major defects in DNA repair or fertility, they show modifications in meiotic crossover recombination patterns. Specifically, the absence of SRS2 shifts the balance between Class I and Class II crossovers, leading to an increase in Class I crossovers and a decrease in MUS81-dependent Class II crossovers. These findings provide new insights into the role of SRS2 in meiosis, suggesting that it has a dual role in the regulation of crossover formation.

genetics↗

bioRxiv2024

DNA binding site II is required for RAD51 recombinogenic activity in Arabidopsis thaliana

Homologous recombination is a major pathway for the repair of DNA double strand breaks, essential both to maintain genomic integrity and to generate genetic diversity. Mechanistically, homologous recombination involves the use of a homologous DNA molecule as a template to repair the break. In eukaryotes, the search for and invasion of the homologous DNA molecule is carried out by two recombinases, RAD51 in somatic cells and RAD51 and DMC1 in meiotic cells. During recombination, the recombinases bind overhanging single-stranded DNA ends to form a nucleoprotein filament, which is the active species in promoting DNA invasion and strand exchange. RAD51 and DMC1 carry two major DNA binding sites - essential for nucleofilament formation and DNA strand exchange, respectively. Here, we show that the function of RAD51 DNA binding Site II is conserved in the plant, Arabidopsis. Mutation of three key amino acids in Site II does not affect RAD51 nucleofilament formation but inhibits its recombinogenic activity, analogous to results from studies of the yeast and human proteins. We further confirm that recombinogenic function of RAD51 DNA binding site II is not required for meiotic DSB repair when DMC1 is present. The Arabidopsis RAD51-II3A separation of function mutant shows a dominant negative phenotype, pointing to distinct biochemical properties of eukaryotic RAD51 proteins. Summary blurbThe activity promoted by RAD51 DNA binding site II is not required for nucleofilament formation, but is essential for subsequent invasion and strand exchange activity of RAD51 in Arabidopsis thaliana.

genetics↗

bioRxiv2022

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments. Author SummaryEssential for fertility and responsible for a major part of genetic variation in sexually reproducing species, meiotic recombination establishes the physical linkages between homologous chromosomes which ensure their balanced segregation in the production of gametes. These linkages, or chiasmata, result from DNA strand exchange catalyzed by the RAD51 and DMC1 recombinases and their numbers and distribution are tightly regulated. Essential for maintaining chromosomal integrity in mitotic cells, the strand-exchange activity of RAD51 is downregulated in meiosis, where it plays a supporting role to the activity of DMC1. Notwithstanding considerable attention from the genetics community, precisely why this is done and the mechanisms involved are far from being fully understood. We show here in the plant Arabidopsis that DMC1 can downregulate RAD51 strand-exchange activity and propose that this may be a general mechanism for suppression of RAD51-mediated recombination in meiosis.

genetics↗

bioRxiv2020

RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis.

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Absence of RAD54 in dmc1 mutants leads to meiotic chromosomal fragmentation. The action of RAD54 in meiotic RAD51 activity is thus downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination. Author SummaryHomologous recombination is a universal pathway which repairs broken DNA molecules through the use of homologous DNA templates. It is both essential for maintenance of genome stability and for the generation of genetic diversity through sexual reproduction. A central step of the homologous recombination process is the search for and invasion of a homologous intact DNA sequence that will be used as template. This key step is catalysed by the RAD51 recombinase in somatic cells and RAD51 and DMC1 in meiotic cells, assisted by a number of associated factors. Among these, the chromatin-remodelling protein RAD54 is a required cofactor for RAD51 in mitotic cells. Understanding of its role during meiotic recombination however remains elusive. We show here that RAD54 is required for repair of meiotic double strand breaks by RAD51 in the plant Arabidopsis thaliana, and this function is downstream of the meiotic role of RAD51 in supporting the activity of DMC1. These results provide new insights into the regulation of the central step of homologous recombination in plants and very probably also other multicellular eukaryotes.

genetics↗

bioRxiv2020

DNA polymerase epsilon is a central coordinator of heterochromatin structure and function in Arabidopsis

BackgroundChromatin organizes the DNA molecule and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. ResultsWe isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, release of heterochromatin silencing in POL2A deficient mutants paradoxically occurs in a chromatin context of increased level of these two repressive epigenetic marks. At the nuclear level, POL2A defect is associated with fragmentation of heterochromatin. ConclusionThese results indicate that POL2A is critical to secure both heterochromatin structure and function. We also reveal that unhindered replisome progression is required for the faithful propagation of DNA methylation through the cell cycle.

genomics↗