Biology subjects

Ahmed-Seghir, S.

Publications and source records attributed to Ahmed-Seghir, S..

3 recordsLinked to original sources

bioRxiv2026

A Multiplex Droplet Digital PCR Assay for Chromosome Copy Number Determination in Candida albicans

Chromosome copy number variation (CNV) is a major contributor to genome plasticity and adaptation in Candida albicans, a leading fungal pathogen of humans. Aneuploidy, defined as deviations from the normal diploid chromosome set, rapidly alters gene dosage, enabling tolerance to host-imposed and antifungal stress. Accurate detection and quantification of chromosomal copy number changes are thus essential to dissect the mechanisms by which C. albicans adapts and evolves. Here, we describe the development, optimization, and validation of a six-color, 16-plex droplet digital PCR assay for simultaneous quantification of all C. albicans chromosome arms in a single reaction. Each target is detected by a unique dual-color or single-color combination of probes, enabling high-order multiplexing through binary fluorescence encoding. Following optimization of probe concentrations, PCR cycling parameters, genomic DNA extraction and pre-treatment with restriction enzymes, the assay provides accurate, reproducible chromosome-level copy number estimates that correlate closely with WGS results across euploid and aneuploid isolates. Compared to whole-genome sequencing, the assay is rapid, cost-effective, and scalable, requiring minimal DNA input and allowing high-throughput analysis of large isolate collections. The 16-plex assay thus provides a platform for dissecting genome instability and adaptive evolution in C. albicans. Article SummaryWe developed and validated a 16-plex droplet digital PCR assay that estimates chromosome dosage across the entire genome of the human fungal pathogen C. albicans in a single reaction. The assay uses six fluorescent colors and unique color combinations to track one marker on each chromosome arm, enabling rapid detection of aneuploidy (extra or missing chromosomes). Results closely matched whole-genome sequencing for isolates with simple aneuploid forms and detected low-frequency trisomic clones in mixed populations. With optimized DNA preparation, this method provides a practical tool for screening genome instability in research and clinical settings.

microbiology↗

bioRxiv2025

RNA Transcripts Serve as a Template for Double-Strand Break Repair in Human Cells

Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Pol{zeta}) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.

molecular biology↗

bioRxiv2023

A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells

When replication forks encounter DNA lesions that cause polymerase stalling a checkpoint pathway is activated. The ATR-dependent intra-S checkpoint pathway mediates detection and processing of sites of replication fork stalling to maintain genomic integrity. Several factors involved in the global checkpoint pathway have been identified, but the response to a single replication fork barrier (RFB) is poorly understood. We utilized the E.coli-based Tus-Ter system in human MCF7 cells and showed that the Tus protein binding to TerB sequences creates an efficient site-specific RFB. The single fork RFB was sufficient to activate a local, but not global, ATR-dependent checkpoint response that leads to phosphorylation and accumulation of DNA damage sensor protein {gamma}H2AX, confined locally to within a kilobase of the site of stalling. These data support a model of local management of fork stalling, which allows global replication at sites other than the RFB to continue to progress without delay.

molecular biology↗