Assays for Nonhomologous End Joining in Extracts

Budman, Joe; Chu, Gilbert

doi:10.1016/s0076-6879(06)08027-x

Cited by 9 publications

(10 citation statements)

References 11 publications

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“…Furthermore, in pancreatic cancer cells constructed to stably express an NHEJ reporter that measures repair of an I-SceI-induced DSB by GFP expression (Mao et al, 2009), depletion of FBXW7 by siRNAs significantly inhibited NHEJ (Figures 2F and S2G). Finally, we incubated linearized plasmid DNA (EcoRI, 5′ overhangs) with cell-free extracts (Budman and Chu, 2006) from two FBXW7 isogenic pairs of cells and found reduced end-joining products in FBXW7 −/− HCT116 and DLD1 cells (Figure 2G). Importantly, we found that stable expression of wild type FBXW7α completely rescued the NHEJ defect observed in FBXW7 −/− cells, while FBXW7α S26A mutant had significantly reduced NHEJ activity relative to wild type (Figures 2H and S2H).…”

Section: Resultsmentioning

confidence: 99%

FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4

et al. 2016

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SUMMARY FBXW7 is a haploinsufficient tumor suppressor with loss-of-function mutations occurring in human cancers. FBXW7 inactivation causes genomic instability, yet the mechanism remains elusive. Here we show that FBXW7 facilitates non-homologous end-joining (NHEJ) repair and FBXW7 depletion causes radiosensitization. In response to ionizing radiation, ATM phosphorylates FBXW7 at serine 26 to recruit it to DNA double-strand break (DSB) sites, while activated DNA-PKcs phosphorylates XRCC4 at serines 325/326 which promotes binding of XRCC4 to FBXW7. SCFFBXW7 E3 ligase then promotes polyubiquitylation of XRCC4 at lysine 296 via K63-linkage for enhanced association with the Ku70/80 complex to facilitate NHEJ repair. Consistent with these findings, a small molecule inhibitor that abrogates XRCC4 polyubiquitylation reduces NHEJ repair. Our study demonstrates one mechanism by which FBXW7 contributes to genome integrity and implies that inactivated FBXW7 in human cancers could be a strategy for increasing efficacy of radiotherapy.

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Section: Resultsmentioning

confidence: 99%

FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4

et al. 2016

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“…We prepared DNA substrates for the PCR-based assay as previously described (18,24) and prepared DNA substrates for the gel-based assay by PCR amplification of a 1-kb DNA fragment. Digestion of the PCR product with EcoRI or EcoRV or double digestion with EcoRV and KpnI released a DNA fragment with EcoRI-EcoRI, EcoRV-EcoRV, or EcoRV-KpnI ends, which was resolved by agarose gel electrophoresis and purified from the gel.…”

Section: Methodsmentioning

confidence: 99%

Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends

Tsai

Kim

Chu

2007

Proc. Natl. Acad. Sci. U.S.A.

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165

154

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DNA repair ͉ nonhomologous end-joining ͉ V(D)J recombinationT he nonhomologous end-joining (NHEJ) pathway is conserved in eukaryotes, from yeast to humans. Without requiring homologous DNA, NHEJ repairs DNA double-strand breaks produced by xenobiotic agents, such as topoisomerase II inhibitors and ionizing radiation, or by the cellular pathway for V(D)J recombination of the immunoglobulin genes (1). Even when the structure of the DNA ends prevents ligation, NHEJ processes the ends and repairs the breaks with high efficiency and minimal nucleotide loss. For ligatable ends, such as the blunt signal ends created by V(D)J recombination, NHEJ suppresses processing and repairs the breaks directly. Thus, NHEJ optimizes the preservation of DNA sequence, but the mechanism is not understood.

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“…Extract Preparation-We prepared whole cell extracts as described previously (21)(22)(23)) from pellets of 10-liter suspension cultures of HeLa S3 cells grown at the National Cell Culture Center (Minneapolis, MN) and lymphoblastoid cells (GM00558C, Coriell Cell Repositories) grown in medium consisting of RPMI 1640, 15% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 1% penicillin/streptomycin (Invitrogen). To immunodeplete XRCC4/Ligase IV, we preincubated extract at 4°C for 90 min with XRCC4 antibody (Serotec) at a ratio of 1 l of antibody to 15 l of extract, incubated the mixture with washed protein A-Sepharose beads (Santa Cruz Biotechnology) at 4°C for 90 min with rotation, and centrifuged the mixture at 4000 ϫ g for 5 min, leaving the supernatant for the NHEJ reaction.…”

Section: Methodsmentioning

confidence: 99%

Processing of DNA for Nonhomologous End-joining Is Controlled by Kinase Activity and XRCC4/Ligase IV

Budman

Kim

Chu

2007

Journal of Biological Chemistry

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Nonhomologous end-joining (NHEJ) repairs DNA doublestrand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence. Nonhomologous end-joining (NHEJ)2 preserves chromosomal integrity by repairing DNA double-strand breaks. Processing of DNA ends is a key step in NHEJ since double-strand breaks often create ends that are not directly ligatable. For example, ionizing radiation generates nucleotides with aberrant structures such as 3Ј-phosphate or 3Ј-phosphoglycolate groups, which must be removed by nuclease activity before ligation can occur.Processing of DNA ends in vivo has been studied in the context of V(D)J recombination. The RAG1/RAG2 dimer cleaves two sites in the immunoglobulin locus to create two hairpin coding ends and two blunt signal ends (1). Nuclease activity opens the hairpin ends to leave 3Ј overhangs, which are filled in to generate P-nucleotide addition (2). To fill in 3Ј overhangs, a polymerase must synthesize DNA from a primer on the opposing end. Nucleotide deletion also occurs, but nuclease activity is limited to less than 20 nucleotides, often back to regions of microhomology (3, 4). Strikingly, the blunt signal ends are joined without processing. Thus, the NHEJ reaction is strongly biased toward the preservation of DNA sequence, suppressing processing if it is not needed and limiting the extent of processing when it is required.The NHEJ reaction requires core proteins and processing enzymes (5). The core proteins include Ku, DNA-PKcs, XRCC4/Ligase IV (XL), and Cernunnos (also named XRCC4-like factor, XLF) (6, 7). Proposed processing enzymes include the nuclease Artemis (8) and DNA polymerases and (9 -12). The biochemical properties of these proteins suggest that NHEJ repairs double-strand breaks in an ordered series of steps. Ku binds to DNA ends and translocates inward, recruiting DNAPKcs (13, 14). DNA-PKcs brings the ends together, activating its kinase activity (15). DNA-PKcs then phosphorylates itself and its target proteins (16). The ends are processed if necessary, and XL catalyzes the final ligation step (17).Important questions remain unanswered. NHEJ requires DNA-PKcs kinase activity (18), but it is not known whether the kinase activity aff...

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Assays for Nonhomologous End Joining in Extracts

Cited by 9 publications

References 11 publications

FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4

FBXW7 Facilitates Nonhomologous End-Joining via K63-Linked Polyubiquitylation of XRCC4

Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends

Processing of DNA for Nonhomologous End-joining Is Controlled by Kinase Activity and XRCC4/Ligase IV

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