Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining
In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcs to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV...
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| Format: | Article |
| Language: | English |
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Wiley
2010-01-01
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| Series: | Journal of Nucleic Acids |
| Online Access: | http://dx.doi.org/10.4061/2010/823917 |
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| author | Cynthia L. Hendrickson Shubhadeep Purkayastha Elzbieta Pastwa Ronald D. Neumann Thomas A. Winters |
| author_facet | Cynthia L. Hendrickson Shubhadeep Purkayastha Elzbieta Pastwa Ronald D. Neumann Thomas A. Winters |
| author_sort | Cynthia L. Hendrickson |
| collection | DOAJ |
| description | In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcs to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcs appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcs protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways. |
| format | Article |
| id | doaj-art-00cd3c3a0d2843b5a14bc311c2416c41 |
| institution | Kabale University |
| issn | 2090-021X |
| language | English |
| publishDate | 2010-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Nucleic Acids |
| spelling | doaj-art-00cd3c3a0d2843b5a14bc311c2416c412025-02-03T05:46:19ZengWileyJournal of Nucleic Acids2090-021X2010-01-01201010.4061/2010/823917823917Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End JoiningCynthia L. Hendrickson0Shubhadeep Purkayastha1Elzbieta Pastwa2Ronald D. Neumann3Thomas A. Winters4Radiology & Imaging Sciences Department, Nuclear Medicine Section, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USARadiology & Imaging Sciences Department, Nuclear Medicine Section, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USAMolecular Genetics Department, Medical University of Lodz, Lodz 92-215, PolandRadiology & Imaging Sciences Department, Nuclear Medicine Section, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USARadiology & Imaging Sciences Department, Nuclear Medicine Section, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USAIn mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcs to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcs appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcs protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.http://dx.doi.org/10.4061/2010/823917 |
| spellingShingle | Cynthia L. Hendrickson Shubhadeep Purkayastha Elzbieta Pastwa Ronald D. Neumann Thomas A. Winters Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining Journal of Nucleic Acids |
| title | Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining |
| title_full | Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining |
| title_fullStr | Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining |
| title_full_unstemmed | Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining |
| title_short | Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining |
| title_sort | coincident in vitro analysis of dna pk dependent and independent nonhomologous end joining |
| url | http://dx.doi.org/10.4061/2010/823917 |
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