Background: Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair.
Aim: To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells.
Methods: Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and gamma-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed.
Results: Transfer of chromosome 8 (location of DNA-PKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene.
Conclusion: We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1136/jmg.2009.068866 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways.
bioRxiv
December 2024
Division of Neurobiology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 600 North Wolfe Street, CMSC 5 South, Baltimore, MD21287.
Huntington's Disease (HD), a progressive neurodegenerative disorder with no disease-modifying therapies, is caused by a CAG repeat expansion in the HD gene encoding polyglutamine-expanded huntingtin (HTT) protein. Mechanisms of HD cellular pathogenesis and cellular functions of the normal and mutant HTT proteins are still not completely understood. HTT protein has numerous interaction partners, and it likely provides a scaffold for assembly of multiprotein complexes many of which may be altered in HD.
View Article and Find Full Text PDFTargeted knockdown of ATM, ATR, and PDEδ increases Gag HIV-1 VLP production in HEK293 cells.
Appl Microbiol Biotechnol
January 2025
Grup d'Enginyeria de Bioprocessos i Biocatàlisi Aplicada, ENG4BIO, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193, Barcelona, Spain.
Several strategies have been developed in recent years to improve virus-like particle (VLP)-based vaccine production processes. Among these, the metabolic engineering of cell lines has been one of the most promising approaches. Based on previous work and a proteomic analysis of HEK293 cells producing Human Immunodeficiency Virus-1 (HIV-1) Gag VLPs under transient transfection, four proteins susceptible of enhancing VLP production were identified: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ).
View Article and Find Full Text PDFGenome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations.
Nat Biotechnol
November 2024
Department of Biology, Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
Article Synopsis
- The DNA-PKcs inhibitor AZD7648 improves the efficiency of CRISPR-Cas9 gene editing for homology-directed repair, showing promise for clinical use.
- However, the use of AZD7648 may lead to significant unintended outcomes such as large deletions, loss of chromosome arms, and chromosomal rearrangements.
- These large-scale genetic changes may go undetected by standard editing tests, highlighting the need for further investigation into the potential risks associated with using AZD7648 in genome editing.
The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations.
Nat Commun
November 2024
Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.
Article Synopsis
- The study investigates how the surrounding DNA sequence affects the repair of double-stranded breaks caused by CRISPR/Cas9, using various genetically modified mouse embryonic stem cell lines.
- Researchers analyzed over 236,000 mutation outcomes from 2800 synthetic DNA sequences, discovering specific roles of DNA repair proteins like Prkdc and Polm in generating small insertions and deletions.
- They developed predictive models for these mutational outcomes based on their findings, enhancing the understanding of DNA repair mechanisms and enabling more accurate control of CRISPR-induced mutations.
Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.
Nucleic Acids Res
December 2024
Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH 43215, USA.
Article Synopsis
- Researchers developed enhanced single-stranded DNA (esDNA) templates with chemical modifications that significantly improve the efficiency of genome editing when used with Cas9, achieving 2-3 times higher knock-in rates compared to standard ssDNA.
- In specific cell types, such as airway basal stem cells and CD34+ hematopoietic cells, esDNA facilitated correction of target genes (CFTR, HBB, CCR5) in over 50% of cases, indicating strong potential for therapeutic applications.
- However, esDNA wasn't effective in induced pluripotent stem cells due to the lack of the nuclease TREX1, suggesting further research is needed for scalable production of modified ssDNA for gene insertion.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!