Abstract
DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.
Highlights
The genetic stability of a cell is constantly challenged by environmental and endogenous factors. 50,000–100,000 different damage events have been estimated to occur each day to the DNA in a single human cell [1]
The m863 allele was isolated during a mutagenesis screen aimed at identifying factors involved in dopaminergic (DA) neuron differentiation in zebrafish embryos and early larvae, using tyrosine hydroxylase expression as a marker. m863 mutant embryos at 3 days post fertilization had fewer DA neurons in the pretectum and retina, while early differentiating DA neurons of the posterior tuberculum appeared largely normal (Fig 1A–1F)
The pleiotropic m863 mutant phenotype with smaller size of tissues forming from rapidly proliferating cells during early larval development and enhanced cell death suggest that the affected gene may be involved in control of cell survival and proliferation
Summary
The genetic stability of a cell is constantly challenged by environmental and endogenous factors. 50,000–100,000 different damage events have been estimated to occur each day to the DNA in a single human cell [1]. DNA damage has to be uncovered and repaired before or during genome replication to ensure integrity of the genome. Proper cell cycle progression and DNA repair are meticulously controlled by multiple factors including the DNA damage-binding protein (DDB) complex. DDB1 functions in DNAdamage repair via two sub-pathways, global genomic repair (GGR) through a heterodimeric complex of DDB1-DDB2, and transcription-coupled repair (TCR) through the interaction of DDB1 and Cockayne syndrome factor (CSA). The failure of NER may contribute to many diseases, including Down syndrome, Parkinson disease, and Huntington's disease [6]. Other functions of DDB1 beyond its accessory role in DNA repair have been associated with the CUL4 E3 ligase complex
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
