Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

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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 arrethe bush balm 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 kt196 torque converter 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.