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Mechanism of radial chromosome formation in human premature aging syndrome cells

人类早衰综合征细胞放射状染色体形成机制

基本信息

批准号：
10793247
负责人：
Anja-Katrin Bielinsky
金额：
$ 24.22万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10793247
关键词：
Aging Amaze Appearance Applications Grants Belief Bioinformatics Biological Models Bloom Syndrome Bone marrow failure Cell Aging Cell Line Cell physiology Cells Centromere Cessation of life Chromosomal translocation Chromosomes Cisplatin Conscious Cross-Linking Reagents Cytogenetics DNA Crosslinking DNA Crosslinking Agent DNA Damage DNA Double Strand Break DNA Repair DNA crosslink DNA ligase III DNA-Directed DNA Polymerase Data Diagnostic Diagnostic tests Double Strand Break Repair Exposure to Fanconi Anemia pathway Fanconi&apos s Anemia Frequencies Genes Genetic Recombination Genomic DNA Goals Grant Human Human Cell Line Individual Knowledge Lasers Literature Mediating Metaphase Microdissection Mission Mitomycin C Modeling Molecular Molecular Profiling Nonhomologous DNA End Joining Normal Cell Outcome Pathology Pathway interactions Patients Phenotype Polymerase Premature aging syndrome Process Production Protocols documentation Radial Radiation Regulation Reporting Research Personnel Role System Testing Therapeutic United States National Institutes of Health bioinformatics pipeline cancer predisposition chromosome fusion clinically relevant crosslink experimental study gene synthesis helicase insight nanopore new therapeutic target novel repaired response telomere ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Radial chromosomes (hereafter “radials”) have been part of the scientific consciousness for more than 50 years. Radials are operationally defined as fusions involving two or more nonhomologous chromosomes and as having more than one centromere. In normal cells, radials are quite rare, and they almost never form spontaneously. Radials have an important clinical relevance, however, since their appearance is a diagnostic hallmark of the bone marrow failure, cancer predisposition and premature aging syndrome, Fanconi anemia (FA). Interestingly though, even in FA cells, radial chromosomes do not appear spontaneously with high frequency but usually need to be induced by exposure to clinically-relevant DNA crosslinking reagents, such as mitomycin C (MMC) and cisplatin. Despite their diagnostic use for many decades, radial formation is still poorly understood, and the literature is filled with conflicting reports about what is required mechanistically for their production. In our first Specific Aim, we describe the construction of a human cell line that is defective for the radiation sensitive 18 (RAD18) gene. RAD18 encodes an E3 ubiquitin ligase and is required for translesion synthesis (TLS), an error-prone DNA damage tolerance pathway, which is a subpathway in the FA-mediated DNA crosslink repair process. Consistent with this role, RAD18-null human cells generated high levels of MMC- induced radials. Unexpectedly, the absence of DNA ligase III (LIGIII) and DNA polymerase theta (POLQ; both key components of alternative non-homologous end joining; A-EJ)) dramatically reduced RAD18-dependent, MMC-induced radial formation. Thus, the radials generated by the absence of RAD18 appear to require the A- EJ pathway. We propose to confirm these observations and extend the generality of these studies by determining the requirement for A-EJ in MMC-induced radial formation in Bloom syndrome (BLM) (another cancer predisposition and premature aging syndrome) and FA-defective cells. Amazingly, there is not a single report in the literature regarding the molecular makeup of a radial chromosome fusion junction. This is unfortunate, since an analysis of fusion junctions for a wide variety of other types of translocations (e.g., canonical reciprocal chromosomal translocations and telomere fusions) has provide a wealth of data regarding the molecular signatures of such junctions as well as insight into the repair mechanisms responsible for their formation. In our Specific Aim 2, we propose to rectify this egregious omission. Specifically, we describe a protocol (Radial-Seq) for isolating, sequencing and bioinformatically analyzing radial chromosome fusions. We expect this analysis to confirm the formation of such junctions by A-EJ and to greatly extend our understanding of the molecular makeup of a radial fusion. In toto, these experiments should mechanistically define the requirements for radial formation which should benefit basic researchers; in addition, the assignment of A-EJ for their formation may help clinicians therapeutically hinder their formation.

项目总结