Taylor EM , Cecillon SM , Bonis A , Chapman JR , Povirk LF , Lindsay HD .

BB\E015662/1 Biotechnology and Biological Sciences Research Council , CA40615 NCI NIH HHS , Medical Research Council , R01 CA040615-25 NCI NIH HHS , BB/E015662/1 Biotechnology and Biological Sciences Research Council , R01 CA040615 NCI NIH HHS , BB_BB/E015662/1 Biotechnology and Biological Sciences Research Council

	Figure 1. Immunodepletion of the MRN complex and Ku70 from X. laevis egg extract. (A) Xenopus laevis egg extract was analysed by Western blotting with antisera against XMre11, XNbs1 and XRad50. (B) Extract was subjected to three rounds of depletion using protein A sepharose coupled to non-specific rabbit IgGs (Δ Mock), anti-XMre11 antibodies (Δ XMre11), anti-XNbs1 antibodies (Δ XNbs1) or anti-XRad50 antibodies (Δ XRad50) or using protein G sepharose coupled to antibodies against Ku70 (Δ XKu70). Double depletion of XMre11 and XKu70 was achieved by two rounds of depletion with α-Ku70 beads followed by one round with α-XMre11 beads. Depletion efficiency was analysed by Western blotting with the appropriate antisera. XChk1 was used as a loading control. A non-specific band is denoted by asterisk.
	Figure 2. NHEJ in XMRN-depleted extracts. (A) Southern blot analysis of NHEJ products generated by incubation of linearized pUC19 template (1 ng/μl) in the indicated egg extracts. Repair products were detected using a fluorescein-labelled pUC19 probe. LMs were converted into MFs, linear dimers (LDs) and closed circular DNA monomers (CC). (B) Agarose gel analysis of repair products resulting from the incubation of radio-labelled pSV56 linear substrates with defined termini in mock-depleted (Δ Mock) or XMre11-depleted (Δ XMre11) extract. Left panel shows end joining of 3′-OH ends. Right panel shows repair of 3′-PG termini. (C) Quantification of DNA forms after 6 h end joining reactions using pSV56 defined linear substrates. The amount of each DNA form is expressed as a percentage of the total signal for each individual lane.
	Figure 3. Analysis of DNA end joining at the nucleotide level. (A) Formation of accurate end joining products from 3′-OH or 3′-PG end substrates during NHEJ reactions. Following the removal of the 3′-PG moeity (•), the 3′-terminal CG residues align and the one-base gap opposite A is filled in by a DNA polymerase. Ligation of the ends generates accurate end joined products which, following treatment with the restriction enzymes TaqαI and BstXI, can be observed on a sequencing gel as 42-bp oligomers. (B) Sequencing gel analysis of NHEJ in X. laevis egg extracts using 3′-OH- and 3′-PG-defined substrates. pSV56 linear substrates with either 3′-OH or 3′-PG ends (1 ng/μl) were incubated in mock-depleted (Δ Mock) or XMre11-depleted (Δ XMre11) extract at 21°C for the indicated times. DNA was digested with TaqαI and BstXI, then resolved on a 20% denaturing polyacrylamide gel. (C) Linearized pSV56 with 3′-OH termini was incubated at 1 ng/μl in undepleted, mock-depleted and XMre11-, XNbs1- or XRad50-depleted extracts for 6 h at 21°C before digestion and analysis as described above. Substrate added to mock-depleted extract at 1 ng/μl and processed immediately serves as a control (0 h). (D) Linearized pSV56 with 3′-OH ends was incubated at 1 ng/μl in X. laevis egg extract at 21°C for the indicated times. Accurate repair occurs at earlier time points than resection-based repair.
	Figure 4. Resection-based end joining is Ku70 independent and occurs at sites of microhomology. (A) pSV56 3′-OH substrate was incubated in mock-, XMre11-, Ku70- and XMre11/Ku70 double-depleted extracts for 6 h. DMSO was added to a final concentration of 0.4% and NU7441 was added to a final concentration of 8 µM as indicated. (B) pSV56 3′-OH substrate was incubated in mock- or XMre11-depleted extract at 21°C for 6 h. DNA was cut with TaqαI and BstXI then further digested with BsaHI or MluI as indicated. (C) Alignment of the 3′-terminal CG residues, dTTP fill-in and ligation generates a BsaHI restriction site (5′-GRCGYC-3′) 12-bp downstream of the TaqαI site in the accurately repaired 42-mer. The 3′–5′ resection of 7 bp on either side of the DNA break creates 4-bp CGCG overhangs that anneal and are ligated without a requirement for further synthesis to yield the 34-mer resected end joining product. A diagnostic MluI restriction site (ACGCGT) is generated 7-bp downstream of the TaqαI site by repair in this manner.

Ahmad, ERCC1-XPF endonuclease facilitates DNA double-strand break repair. 2008, Pubmed

Ahmad, ERCC1-XPF endonuclease facilitates DNA double-strand break repair. 2008, Pubmed
Ahnesorg, XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. 2006, Pubmed
Aoufouchi, Post-translational activation of non-homologous DNA end-joining in Xenopus oocyte extracts. 1997, Pubmed , Xenbase
Audebert, Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. 2004, Pubmed
Bennardo, Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. 2008, Pubmed
Bentley, DNA double strand break repair in human bladder cancer is error prone and involves microhomology-associated end-joining. 2004, Pubmed
Boulton, Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. 1998, Pubmed
Buck, Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. 2006, Pubmed
Calsou, The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA. 1999, Pubmed
Chen, Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair. 2008, Pubmed
Chen, Homogeneous preparations of 3'-phosphoglycolate-terminated oligodeoxynucleotides from bleomycin-treated DNA as verified by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. 2001, Pubmed
Chen, Accurate in vitro end joining of a DNA double strand break with partially cohesive 3'-overhangs and 3'-phosphoglycolate termini: effect of Ku on repair fidelity. 2001, Pubmed , Xenbase
Cheong, DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro. 1999, Pubmed
Corneo, Rag mutations reveal robust alternative end joining. 2007, Pubmed
Costanzo, Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. 2001, Pubmed , Xenbase
D'Amours, The Mre11 complex: at the crossroads of dna repair and checkpoint signalling. 2002, Pubmed
Deriano, Roles for NBS1 in alternative nonhomologous end-joining of V(D)J recombination intermediates. 2009, Pubmed
Di Virgilio, Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11. 2005, Pubmed , Xenbase
Difilippantonio, Distinct domains in Nbs1 regulate irradiation-induced checkpoints and apoptosis. 2007, Pubmed
Dynan, Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. 1998, Pubmed
Felix, A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. 1989, Pubmed , Xenbase
Girard, Radiosensitivity in Nijmegen Breakage Syndrome cells is attributable to a repair defect and not cell cycle checkpoint defects. 2000, Pubmed
Grawunder, Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. 1997, Pubmed
Gu, End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini. 1996, Pubmed , Xenbase
Guirouilh-Barbat, Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends. 2007, Pubmed
Guirouilh-Barbat, Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. 2004, Pubmed
Göttlich, Rejoining of DNA double-strand breaks in vitro by single-strand annealing. 1998, Pubmed , Xenbase
Harfst, Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome. 2000, Pubmed
Howlett, Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients. 2006, Pubmed
Huang, Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction. 2002, Pubmed
Jazayeri, ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. 2006, Pubmed
Kabotyanski, Double-strand break repair in Ku86- and XRCC4-deficient cells. 1998, Pubmed
Khanna, DNA double-strand breaks: signaling, repair and the cancer connection. 2001, Pubmed
Kracker, Nibrin functions in Ig class-switch recombination. 2005, Pubmed
Kuhfittig-Kulle, The mutagenic potential of non-homologous end joining in the absence of the NHEJ core factors Ku70/80, DNA-PKcs and XRCC4-LigIV. 2007, Pubmed
Labhart, Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts. 1999, Pubmed , Xenbase
Leahy, Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. 2004, Pubmed
Lee, Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining. 2007, Pubmed
Lee, Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts. 2004, Pubmed
Liang, Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks. 2008, Pubmed , Xenbase
Liang, Modulation of DNA end joining by nuclear proteins. 2005, Pubmed
Lieber, The mechanism of vertebrate nonhomologous DNA end joining and its role in V(D)J recombination. 2004, Pubmed
Luo, Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. 1999, Pubmed
Lähdesmäki, Delineation of the role of the Mre11 complex in class switch recombination. 2004, Pubmed
Ma, Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. 2002, Pubmed
Ma, Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. 2003, Pubmed
Ma, A biochemically defined system for mammalian nonhomologous DNA end joining. 2004, Pubmed
Manolis, Novel functional requirements for non-homologous DNA end joining in Schizosaccharomyces pombe. 2001, Pubmed
McVey, MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. 2008, Pubmed
Nevaldine, The scid defect results in much slower repair of DNA double-strand breaks but not high levels of residual breaks. 1997, Pubmed
Nick McElhinny, A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. 2005, Pubmed
Pan-Hammarström, Impact of DNA ligase IV on nonhomologous end joining pathways during class switch recombination in human cells. 2005, Pubmed
Paull, A mechanistic basis for Mre11-directed DNA joining at microhomologies. 2000, Pubmed
Pfeiffer, Joining of nonhomologous DNA double strand breaks in vitro. 1988, Pubmed , Xenbase
Reina-San-Martin, Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1. 2005, Pubmed
Riballo, A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci. 2004, Pubmed
Rothkamm, Pathways of DNA double-strand break repair during the mammalian cell cycle. 2003, Pubmed
Sartori, Human CtIP promotes DNA end resection. 2007, Pubmed
Soulas-Sprauel, Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination. 2007, Pubmed
Stewart, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. 1999, Pubmed
Stracker, The carboxy terminus of NBS1 is required for induction of apoptosis by the MRE11 complex. 2007, Pubmed
Stracker, The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together. 2004, Pubmed
Symington, Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. 2002, Pubmed
Tauchi, Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. 2002, Pubmed
Tsukuda, Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. 2005, Pubmed
Varon, Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. 1998, Pubmed
Wang, Biochemical evidence for Ku-independent backup pathways of NHEJ. 2003, Pubmed
West, Molecular views of recombination proteins and their control. 2003, Pubmed
Williams, Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. 2007, Pubmed
Xiao, Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. 1997, Pubmed
Yamaguchi-Iwai, Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells. 1999, Pubmed
Yan, IgH class switching and translocations use a robust non-classical end-joining pathway. 2007, Pubmed
Yang, Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks. 2006, Pubmed
Yeo, V(D)J rearrangement in Nijmegen breakage syndrome. 2000, Pubmed
You, ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. 2005, Pubmed , Xenbase
Yu, Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae. 2003, Pubmed
Yun, CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle. 2009, Pubmed
Zhong, Deficient nonhomologous end-joining activity in cell-free extracts from Brca1-null fibroblasts. 2002, Pubmed
Zhu, Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. 2001, Pubmed
Zhu, Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations. 2002, Pubmed
van Gent, Chromosomal stability and the DNA double-stranded break connection. 2001, Pubmed