Zhu S and Peng A (2016), Non-homologous end joining repair in Xenopus eg...

XB-ART-52515

Sci Rep 2016 Jun 21;6:27797. doi: 10.1038/srep27797.

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Non-homologous end joining repair in Xenopus egg extract.

Zhu S , Peng A .

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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.

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Species referenced: Xenopus
Genes referenced: atm atr

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	Figure 1. Repair of DSB substrates in Xenopus egg extract.(A) Schematic diagram of the DSB repair assay. As described in Materials and Methods, linearized plasmid DNA was generated and incubated in Xenopus egg extract. Plasmid DNA was then re-isolated from egg extracts, and transformed into bacteria cells. The final repair products were isolated from bacteria colonies and subjected to sequencing analysis. (B) As in panel A, incubation of plasmid DNA linearized with Xho1 and Kpn1 in Xenopus egg extracts led to formation of colonies after bacteria transformation. As a control, mixing the linearized plasmid with egg extracts without incubation did not yield colony formation. (C) As in panel A, the linearized plasmid DNA was incubated in Xenopus egg extracts for 0.5 or 2âhr, re-isolated, and transformed into bacteria cells. The repair activity was measured by colony numbers. (D) The repair assay was performed in Xenopus egg extract that was supplemented with or without Ku70 antibody. The repair activity (in relative to the control extract) was measured by colony numbers. (E) The repair assay was performed in Xenopus egg extract that was supplemented with or without DNA-PKcs inhibitor (NU7441). The repair activity was measured by colony formation and compared between extracts with or without the inhibitor. Five types of repair templates were used, as in Fig. S2. In panels CâE, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.05 was considered statistically significant.
	Figure 2. The repair of compatible ends required DNA-PK but not ATM.(A) The pMBPII plasmid was digested by XhoI to generate compatible DSB ends. The DNA substrate was incubated in Xenopus egg extracts, re-isolated, and transformed into bacteria cells. The final repair products were isolated from bacteria colonies and subjected to sequencing analysis. The repair product with large-fragment deletion (200âbp-2âkb) was determined by agarose gel electrophoresis (data not shown). (B) The repair assay was established as in panel A. The extract was supplemented with or without DNA-PKcs inhibitor NU7441. The repair activity (in relative to the control extract) was measured by colony numbers. (C) The repair assay was established as in panel A. The extract was supplemented with or without ATM inhibitor KU60019. The repair activity (in relative to the control extract) was measured by colony numbers. (D) The specificity of ATM and DNA-PKcs inhibitors were confirmed by immunoblotting. Xenopus egg extract were treated with double-stranded oligonucleotides and specific inhibitors as indicated. After incubation for 30âmin at room temperature, samples were analyzed using specific antibodies as indicated. In panels B & C, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.05 was considered statistically significant.
	Figure 3. ATM is required for the repair of non-compatible DSB ends.(A) Xenopus egg extracts were supplemented with or without ATM inhibitor KU60019, and incubated with various types of NHEJ templates as in Fig. S2. The repair activity (in relative to the control extract) was measured by colony numbers. (B) Xenopus egg extracts were supplemented with or without ATM inhibitor KU55933, and incubated with the 5â²/3â² NHEJ template. The repair activity (in relative to the control extract) was measured by colony numbers. (C) Xenopus egg extracts were supplemented with or without caffeine to inhibit ATM/ATR, and incubated with the 5â²/3â² NHEJ template. The repair activity (in relative to the control extract) was measured by colony numbers. (D,E) ATM was depleted fromXenopus egg extract as described in Materials and Methods. The extract was incubated with the 5â²/3â² NHEJ template, and the repair activity (in relative to the control extract) was measured by colony numbers. The efficiency of ATM depletion was confirmed by immunoblotting. (F) Xenopus egg extracts were supplemented with or without ATR inhibitor VE821, and incubated with the 5â²/3â² template. The repair activity (in relative to the control extract) was measured by colony numbers. (H,G) ATR was depleted fromXenopus egg extract. The extract was incubated with the 5â²/3â² NHEJ template, and the repair activity (in relative to the control extract) was measured by colony numbers. The efficiency of ATR depletion was confirmed by immunoblotting. In panels A, B, C, D, F & G, a minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-valueâ<â0.05 was considered statistically significant.
	Figure 4. Processing of non-compatible ends is flexible and specific to the structure of DSB ends.Various NHEJ repair templates with non-compatible ends, as in Fig. S2, were incubated in Xenopus egg extracts, re-isolated, and transformed into bacteria cells. Final repair products were isolated and subjected to sequencing analysis. The NHEJ templates include: blunt/3â²-overhang (A), blunt with 5â²-overhang (B), 3â²-overhang/5â²-overhang (C), 3â²-overhang/3â²-overhang (D), and 5â²-overhang/5â²-overhang (E). The repair assay was performed with or without ATM inhibitor as in Fig. 3B. In each reaction, approximately 15â20 final repair products were sequenced and shown. Nucleotides deleted during DNA repair were indicated by empty triangles. Repair products with large-fragment deletion (200âbp-2âkb) were determined by agarose gel electrophoresis (data not shown).
	Figure 5. End dephosphorylation reduced the repair efficiency without promoting end resection.(A) The 3â²-overhang/5â²-overhang template was incubated with calf intestinal alkaline phosphatase (CIP), or control buffer, prior to the addition in Xenopus egg extracts. The repair activity (in relative to the control extract) was measured by colony numbers. A minimum of three experiments were carried out and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired 2-tailed Studentâs t-test. A p-value0.05 was considered statistically significant. (B) The repair assay with CIP incubation was established as in panel A. Ten final repair products were sequenced and shown. Repair products with large-fragment deletion were determined by agarose gel electrophoresis.
	Figure 6. The pattern of NHEJ end processing is specific to the end structure and dependent on ATM and DNA-PKcs.Compatible ends, and non-compatible ends with blunt/3â²-overhang, blunt/5â²-overhang, and 3â²-overhang/5â²-overhang were predominantly repaired with fill-in and ligation without resection of end nucleotides. In contrast, 3â²-overhang/3â²-overhang or 5â²-overhang/5â²-overhang templates were processed by resection of 3â5 bases and 1â4 base fill-in 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. Direct ligation of compatible ends is dependent on the kinase activity of DNA-PKcs but not ATM. ATM was required for the efficient repair of all non-compatible ends, suggesting that ATM-dependent phosphorylation may regulate both end resection and fill-in.

References [+] :

Budman, Processing of DNA for nonhomologous end-joining by cell-free extract. 2005, Pubmed