Biology subjects

White, C. I.

Publications and source records attributed to White, C. I..

8 recordsLinked to original sources

bioRxiv2024

Context effects on repair of 5'-overhang DSB induced by Cas12a in Arabidopsis.

Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much-studied Cas9 nuclease cleaves DNA to generate blunt-ended, double-strand breaks (DSB), but relatively less is known about the repair of other types of breaks, such as those with 5-overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by End-Joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSB generate deletions at one or both cut-sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single-cut and double-cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of an alternative class of repair events, corresponding to highly efficient repair by Single-strand Annealing recombination.

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↗

bioRxiv2024

Meiotic DSB repair DNA synthesis tracts in Arabidopsis thaliana

We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with [~]400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair recombination. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites, colocalises with the EdU foci. It is currently estimated that [~]10 cross-overs (CO) and an equivalent number of non-cross-overs (NCO) occur in each Arabidopsis male meiosis. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes. Author SummarySexual reproduction involves the fusion of two cells, one from each parent. To maintain a stable chromosome complement across generations, these specialized reproductive cells must be produced through a specialized cell division called meiosis. Meiosis halves the chromosome complement of gametes and recombines the parental genetic contributions in each gamete, generating the genetic variation that drives evolution. The complex mechanisms of meiotic recombination have been intensely studied for many years and we now know that it involves the repair of programmed chromosomal breaks through recombination with intact template DNA sequences on another chromatid. At the molecular level, this is known to involve new DNA synthesis at the sites of repair/recombination and we report here the successful identification and characterisation of this DNA neo-synthesis during meiosis in the flowering plant, Arabidopsis. Both the characteristics and numbers of these DNA synthesis tracts accord with expectations from theory and earlier studies. Potentially applicable to studies in many organisms, this approach provides indelible footprints in the chromosomes and has the great advantage of freeing researchers from dependence on indirect methods involving detection of proteins involved in these dynamic processes.

genetics↗

bioRxiv2024

Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types

O_LIThroughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adjust to the changing environment that can elicit varying amounts of DNA damage. C_LIO_LIUsing a combination of state-of-the-art cell biology methods including whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH), allowing detection of individual mRNA molecules in intact plant tissue and plant cell cycle reporter lines we investigated how the transcriptional activation of a key homologous recombination (HR) gene, RAD51, occurs in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. C_LIO_LIThe results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. C_LIO_LIThis work advances the current comprehension of DNA damage response in plants showing quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants. C_LI

plant biology↗

bioRxiv2022

Quisinostat is a brain-penetrant radiosensitizer in glioblastoma

In recent years, histone deacetylase inhibitors (HDACi) have garnered considerable interest for the treatment of adult and pediatric malignant brain tumors. However, owing to their broad-spectrum nature and inability to effectively penetrate the blood-brain barrier, HDACi have failed to provide significant clinical benefit to glioblastoma (GBM) patients to date. Moreover, global inhibition of HDACs results in widespread toxicity, highlighting the need for selective isoform targeting. While no isoform-specific HDACi are currently available, the second-generation hydroxamic acid-based HDACi quisinostat possesses sub-nanomolar specificity for class I HDAC isoforms, particularly HDAC1 and 2. Recently, we demonstrated that HDAC1 is the essential HDAC in GBM. Here, we provide the first report on the neuro-pharmacokinetic, pharmacodynamic and radiation-sensitizing properties of quisinostat in preclinical models of GBM. We demonstrate that quisinostat is a well-tolerated and brain-penetrant molecule that significantly extends survival when administered in combination with radiation in vivo. The pharmacokinetic-pharmacodynamic-efficacy relationship was established by correlating free drug concentrations and evidence of target modulation in the brain with survival benefit. Together, these data provide a strong rationale for clinical development of quisinostat as a radiosensitizer for the treatment of GBM.

cancer biology↗

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↗