Biology subjects

Davo-Martinez, C.

Publications and source records attributed to Davo-Martinez, C..

4 recordsLinked to original sources

bioRxiv2026

Linking live-cell imaging to single-cell sequencing using genetic barcoding

Linking cellular phenotype to genotype and gene expression is a central goal in biomedical research. Live-cell imaging captures dynamic cellular phenotypes, whereas single-cell sequencing measures genome-wide molecular states, but pairing these readouts for the same cell remains challenging. Here we present Waldo, a genetically encoded single-cell barcoding strategy that can be read out by both live-cell imaging and single-cell sequencing to connect single cells across both modalities. Waldo barcodes are generated by combinatorial expression of synthetic cell-surface receptors, each detected by a cognate peptide probe coupled either to a fluorescent dye or a DNA barcode. Iterative fluorescent probe staining achieves large barcode diversity, while preserving fluorescence channels for phenotypic reporters. As a proof-of-principle, we use Waldo to link cell division phenotypes after chromosome missegregation to the karyotype and transcriptome of the same cells. Waldo provides a general strategy for connecting imaging phenotypes to genome-wide sequencing readouts in single cells.

cell biology↗

bioRxiv2024

Regulation of XPG-DNA damage binding dynamics by pre- and post-incision nucleotide excision repair factors and EXO1

The XPG endonuclease plays a crucial role in nucleotide excision repair (NER) and other genome maintenance pathways. Precise regulation of XPG recruitment and activity during DNA repair is essential to avoid erroneous DNA incisions and genomic instability. In this study, we employed live-cell imaging to investigate how XPG function is regulated during NER, focusing on its dynamic interactions with key factors involved in the pre- and post-incision steps. We found that TFIIH and XPA facilitate the recruitment and association of XPG with DNA damage, and that XPG localizes separately from TFIIH to UV-induced lesions. Furthermore, our results show that XPGs dissociation from DNA damage is triggered by its own incision activity as well as by that of XPF. Additionally, the exonuclease EXO1 promotes XPG dissociation, likely by processing incised DNA, even in the absence of XPG-mediated incision. Our findings help to better understand the regulatory mechanisms that control XPG activity during NER and provide important insights into the complex dynamics of the repair process.

genetics↗

bioRxiv2023

Recovery of protein synthesis to measure transcription-coupled DNA repair in living cells and tissues

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that protects against the negative effects of transcription-blocking DNA lesions. Hereditary TC-NER deficiency causes pleiotropic and often severe neurodegenerative and progeroid symptoms. Multiple assays have been developed for the clinic and for research to measure TC-NER activity, which is hampered by the relatively low abundance of repair events taking place in transcribed DNA. Recovery of RNA Synthesis is widely used as indirect TC-NER assay based on the notion that lesion-blocked transcription only resumes after successful TC-NER. Here, we show that measuring novel synthesis of a protein that has been degraded prior to DNA damage induction is an equally effective but more versatile manner to indirectly monitor TC-NER. This Recovery of Protein Synthesis (RPS) assay is readily adaptable for use with different degradable proteins and readouts, including fluorescence imaging and immunoblot. Moreover, with the RPS assay TC-NER activity can be measured in real-time, in various living cells types and even in differentiated tissues of living organisms. As example, we show that TC-NER capacity declines in aging muscle tissue of C. elegans. Therefore, the RPS assay constitutes an important novel clinical and research tool to investigate transcription-coupled DNA repair.

genetics↗

bioRxiv2021

C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair

The 10-subunit TFIIH complex is vital to both transcription initiation and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental brittle hair disorder trichothiodystrophy (TTD). Some TTD features are thought to be caused by subtle transcription or gene expression defects. Strikingly, TTDA/GTF2H5 knockout mice are not viable, which makes it difficult to investigate how TTDA/GTF2H5 promotes transcription in vivo. Here, we show that deficiency of the C. elegans TTDA ortholog GTF-2H5 is, however, compatible with viability and growth, in contrast to depletion of other TFIIH subunits. We also show that GTF-2H5 promotes the stability of TFIIH in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of the TFIIH complex to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in the absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as potential model for studying the pathogenesis of this disease.

genetics↗