Biology subjects

Doksani, Y.

Publications and source records attributed to Doksani, Y..

6 recordsLinked to original sources

bioRxiv2026

DNA2Graph enables automated identification of non-linear DNA molecules in electron microscopy

Electron microscopy of spread and rotary-shadowed DNA molecules provides a direct readout of DNA structure and remains uniquely informative for studying replication, recombination and repair intermediates. However, quantitative EM analysis is limited by the rarity of biologically informative structures and by the time required for expert operators to inspect very large numbers of molecules. Automated image acquisition and stitching have increased the scale of EM datasets but have shifted the main bottleneck from image collection to image analysis. Standard image segmentation tools and machine-learning approaches fail to faithfully preserve molecular continuity in EM images of DNA molecules contrasted by rotary shadowing. Here we present DNA2Graph, an open-source software for segmentation of DNA molecules from EM images. Rather than treating segmentation as a purely pixel-level task, DNA2Graph represents each molecule as a spatial graph of nodes and edges. It applies dedicated graph-based error-correction algorithms that repair signal interruptions and spurious connections introduced during segmentation, while enforcing the biological priors of continuity and thinness. DNA2Graph classifies molecules as linear or non-linear, thereby converting large imaging datasets into focused lists of candidate structures for operator review. We validated DNA2Graph on two genomic DNA datasets: a structure-poor, non-enriched human sample and a structure-rich yeast sample. DNA2Graph reduced the number of molecules requiring operator review by 45-fold and 7-fold, respectively. In addition, DNA2Graph-assisted review slightly improved the operators structure-detection sensitivity compared to unaided image review. The software also enables automated length measurement of individual DNA molecules and generates machine-readable outputs for downstream quantitative and computational analyses. DNA2Graph does not require training on manually annotated data, can run on a personal computer and can be adapted across experimental preparations through interpretable parameters.

molecular biology↗

bioRxiv2025

Inhibition of lagging strand replication by G-rich telomeric DNA and the shelterin subunit POT1

Telomeres preserve stable eukaryotic chromosomes by protecting the natural chromosome ends from DNA repair but pose a persistent challenge to the replication machinery and an endogenous source of replication stress. Different features have been implicated in causing this effect but how the canonical replication process is altered at telomeres in mechanistic terms remains poorly understood. To address this question, we have reconstituted telomere replication with purified human proteins. This system reveals that while G-rich telomeric DNA directly and specifically blocks lagging strand replication in a manner counteracted by BLM helicase, shelterin unexpectedly acts as an additional lagging strand barrier. Biochemical experiments and electron microscopy imaging show that POT1-containing shelterin complexes induce Okazaki fragment skipping by binding the lagging strand template, generating large single-stranded gaps that remain unreplicated. Our study defines how the core components of telomeres interfere with the canonical replication process, identifying several potential sources of replication stress.

molecular biology↗

bioRxiv2025

RNase H1 counteracts DNA damage and ameliorates SMN-dependent phenotypes in a Drosophila model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is caused by a deficiency of the Survival Motor Neuron (SMN) protein. Mutations in SMN disrupt mRNA splicing and translation, leading to maladaptive changes in transcriptomes, proteomes, neuroinflammation, and metabolism, which drive motor neuron degeneration in SMA patients. Using a Drosophila SMA model, we found that systemic depletion of Smn leads to accumulation of RNA:DNA hybrids (R-loops), increased DNA damage, dysregulation of amino acids and sugar metabolism and activation of the innate immune response, recapitulating key pathological features reported in mammalian models and severe SMA patients. Persistent DNA damage in Smn-deficient flies alters cell proliferation rates in larval brains and induces extensive cell death in the developing eye. Importantly here, we show that stimulating the resolution of RNA:DNA hybrids with transgenic human RNAse H1 prevents the accumulation of DNA damage and attenuates the transcriptome and amino acid alterations induced by Smn depletion, mitigating the Smn-dependent cellular and developmental abnormalities, in Smn-deficient flies. Our data suggest that depletion of Smn causes an accumulation of aberrant transcripts and chronic DNA damage, which--along with the altered metabolomic profiles associated with Smn deficiency--trigger systemic inflammatory responses, ultimately affecting neuronal function and survival.

genetics↗

bioRxiv2022

Molecular origins of genome instability following a single chromosome mis-segregation event

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures were upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

cell biology↗

bioRxiv2021

A method to enrich and purify centromeric DNA from human cells

Centromeres are key elements for chromosome segregation. Canonical centromeres are built over long-stretches of tandem repetitive arrays. Despite being quite abundant compared to other loci, centromere sequences overall still represent only 2 to 5% of the human genome, therefore studying their genetic and epigenetic features is a major challenge. Furthermore, sequencing of centromeric regions requires high coverage to fully analyze length and sequence variations, which can be extremely costly. To bypass these issues, we have developed a technique based on selective restriction digestion and size fractionation to enrich for centromeric DNA from human cells. Combining enzymes capable of cutting at high frequency throughout the genome, except within most human centromeres, with size-selection of >20 kb fragments resulted in over 25-fold enrichment in centromeric DNA. Sequencing of the enriched fractions revealed that up to 60% of the enriched material is made of centromeric DNA. This approach has great potential for making sequencing of centromeric DNA more affordable and efficient and for single DNA molecule studies.

molecular biology↗

bioRxiv2020

Telomere damage induces internal loops that generate telomeric circles

Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops), that occur in proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage with the accumulation of extrachromosomal telomeric circles and telomere erosion.

molecular biology↗