Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells
Abstract Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to all...
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Nature Portfolio
2024-11-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-54545-y |
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| author | Mariia Mikhova Noah J. Goff Tomáš Janovič Joshua R. Heyza Katheryn Meek Jens C. Schmidt |
| author_facet | Mariia Mikhova Noah J. Goff Tomáš Janovič Joshua R. Heyza Katheryn Meek Jens C. Schmidt |
| author_sort | Mariia Mikhova |
| collection | DOAJ |
| description | Abstract Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115 Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop an approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and allowed us to estimate the overall repair capacity of the NHEJ pathway in living cells. |
| format | Article |
| id | doaj-art-762942600f0844a6ad8f1db2cc791d1d |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-762942600f0844a6ad8f1db2cc791d1d2024-11-24T12:32:41ZengNature PortfolioNature Communications2041-17232024-11-0115111710.1038/s41467-024-54545-ySingle-molecule imaging reveals the kinetics of non-homologous end-joining in living cellsMariia Mikhova0Noah J. Goff1Tomáš Janovič2Joshua R. Heyza3Katheryn Meek4Jens C. Schmidt5Department of Biochemistry and Molecular Biology, Michigan State UniversityDepartment of Microbiology, Genetics, and Immunology, Michigan State UniversityInstitute for Quantitative Health Science and Engineering, Michigan State UniversityInstitute for Quantitative Health Science and Engineering, Michigan State UniversityDepartment of Microbiology, Genetics, and Immunology, Michigan State UniversityInstitute for Quantitative Health Science and Engineering, Michigan State UniversityAbstract Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115 Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop an approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and allowed us to estimate the overall repair capacity of the NHEJ pathway in living cells.https://doi.org/10.1038/s41467-024-54545-y |
| spellingShingle | Mariia Mikhova Noah J. Goff Tomáš Janovič Joshua R. Heyza Katheryn Meek Jens C. Schmidt Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells Nature Communications |
| title | Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells |
| title_full | Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells |
| title_fullStr | Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells |
| title_full_unstemmed | Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells |
| title_short | Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells |
| title_sort | single molecule imaging reveals the kinetics of non homologous end joining in living cells |
| url | https://doi.org/10.1038/s41467-024-54545-y |
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