PMID:27324260

Citation	Zhu, S and Peng, A (2016) Non-homologous end joining repair in Xenopus egg extract. Sci Rep 6:27797
Abstract	Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair mechanism. We characterized here a series of plasmid-based DSB templates that were repaired in Xenopus egg extracts via the canonical, Ku-dependent NHEJ pathway. We showed that the template with compatible ends was efficiently repaired without end processing, in a manner that required the kinase activity of DNA-PKcs but not ATM. Moreover, non-compatible ends with blunt/3'-overhang, blunt/5'-overhang, and 3'-overhang/5'-overhang were predominantly repaired with fill-in and ligation without the removal of end nucleotides. In contrast, 3'-overhang/3'-overhang and 5'-overhang/5'-overhang templates were processed by resection of 3-5 bases and fill-in of 1-4 bases prior to end ligation. Therefore, the NHEJ machinery exhibited a strong preference for precise repair; the presence of neither non-compatible ends nor protruding single strand DNA sufficiently warranted the action of nucleases. ATM was required for the efficient repair of all non-compatible ends including those repaired without end processing by nucleases, suggesting its role beyond phosphorylation and regulation of Artemis. Finally, dephosphorylation of the 5'-overhang/3'-overhang template reduced the efficiency of DNA repair without increasing the risk of end resection, indicating that end protection via prompt end ligation is not the sole mechanism that suppresses the action of nucleases.
Links	PubMed PMC4914968 Online version:10.1038/srep27797
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
XENLA:PRKDC		GO:0006303: double-strand break repair via nonhomologous end joining	ECO:0000314:		P		In figure E1, a multitude of overhang types were tested with and without the inhibition of DNA-PKcs. In all cases, the inhibition of DNA-PKcs significantly reduced the repair activity of the egg extract as compared to the control.	complete CACAO 12482
XENLA:PRKDC		GO:0006303: double-strand break repair via nonhomologous end joining	ECO:0000315:		P		Figure 2B shows how DNA -PKcs is required for efficient non homologous end repair activity, as evidenced with the assay on repair activity with or without DNA-PKcs inhibitor NU7441. When DNA-PKcs was inhibited, the activity of repair of double stranded breaks via NHEJ was significantly decreased. Xenopus laevis.	complete CACAO 12501

References

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