6 research outputs found
TARG1 protects against toxic DNA ADP-ribosylation
ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed 'DarT' from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation
Molecular basis for DarT ADP-ribosylation of a DNA base
ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2–4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6–9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thyminelinked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.Peer reviewedFinal Accepted Versio
CellProfiler plugins -- an easy image analysis platform integration for containers and Python tools
CellProfiler is a widely used software for creating reproducible, reusable
image analysis workflows without needing to code. In addition to the >90
modules that make up the main CellProfiler program, CellProfiler has a plugins
system that allows for creation of new modules which integrate with other
Python tools or tools that are packaged in software containers. The
CellProfiler-plugins repository contains a number of these CellProfiler
modules, especially modules that are experimental and/or dependency-heavy.
Here, we present an upgraded CellProfiler-plugins repository with examples of
accessing containerized tools, improved documentation, and added
citation/reference tools to facilitate the use and contribution of the
community.Comment: 17 pages, 2 figures, 1 tabl
Reproducible image-based profiling with Pycytominer
Technological advances in high-throughput microscopy have facilitated the
acquisition of cell images at a rapid pace, and data pipelines can now extract
and process thousands of image-based features from microscopy images. These
features represent valuable single-cell phenotypes that contain information
about cell state and biological processes. The use of these features for
biological discovery is known as image-based or morphological profiling.
However, these raw features need processing before use and image-based
profiling lacks scalable and reproducible open-source software. Inconsistent
processing across studies makes it difficult to compare datasets and processing
steps, further delaying the development of optimal pipelines, methods, and
analyses. To address these issues, we present Pycytominer, an open-source
software package with a vibrant community that establishes an image-based
profiling standard. Pycytominer has a simple, user-friendly Application
Programming Interface (API) that implements image-based profiling functions for
processing high-dimensional morphological features extracted from microscopy
images of cells. Establishing Pycytominer as a standard image-based profiling
toolkit ensures consistent data processing pipelines with data provenance,
therefore minimizing potential inconsistencies and enabling researchers to
confidently derive accurate conclusions and discover novel insights from their
data, thus driving progress in our field.Comment: 13 pages, 4 figure
The interplay of TARG1 and PARG protects against genomic instability
The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ri-bosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine-and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability
The interplay of TARG1 and PARG protects against genomic instability
Summary: The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ribosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine- and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability