Abstract
The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation. This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining). We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining. In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site. In the other one, the DSB is made by the RAG1 and RAG2 proteins. After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion. We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency. V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use. Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination. These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antigens, Nuclear*
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B-Lymphocytes / drug effects
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B-Lymphocytes / metabolism
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Cell Line / drug effects
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Cell Line / metabolism
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Chromosome Breakage*
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Cricetinae
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Cricetulus
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DNA / genetics
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DNA / metabolism*
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DNA Helicases*
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DNA Ligase ATP
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DNA Ligases / deficiency
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DNA Ligases / physiology
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DNA Nucleotidyltransferases / physiology*
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DNA Repair / physiology*
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DNA-Activated Protein Kinase
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DNA-Binding Proteins / deficiency
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / physiology
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Endonucleases*
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Fibroblasts / drug effects
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Fibroblasts / metabolism
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Gene Rearrangement / physiology*
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Genes, RAG-1
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Homeodomain Proteins / physiology
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Humans
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Ku Autoantigen
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Macromolecular Substances
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Nuclear Proteins / deficiency
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Nuclear Proteins / physiology
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Polymerase Chain Reaction
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Polymorphism, Restriction Fragment Length
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Protein Serine-Threonine Kinases / deficiency
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Protein Serine-Threonine Kinases / physiology
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Proteins / physiology
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Recombination, Genetic / physiology*
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Sequence Homology, Nucleic Acid
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Transfection
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VDJ Recombinases
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X-ray Repair Cross Complementing Protein 1
Substances
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Antigens, Nuclear
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DNA-Binding Proteins
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Homeodomain Proteins
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Macromolecular Substances
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Nuclear Proteins
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Proteins
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RAG2 protein, human
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V(D)J recombination activating protein 2
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X-ray Repair Cross Complementing Protein 1
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X-ray repair cross complementing protein 3
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XRCC4 protein, human
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XRCC8 protein, human
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xeroderma pigmentosum group F protein
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RAG-1 protein
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DNA
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DNA-Activated Protein Kinase
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PRKDC protein, human
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Protein Serine-Threonine Kinases
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DNA Nucleotidyltransferases
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VDJ Recombinases
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ERCC1 protein, human
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Endonucleases
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DNA Helicases
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XRCC5 protein, human
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Xrcc6 protein, human
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Ku Autoantigen
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DNA Ligases
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DNA Ligase ATP