Processing of DNA for nonhomologous end‐joining by cell‐free extract

In mammalian cells, nonhomologous end‐joining (NHEJ) repairs DNA double‐strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell‐free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA‐PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of opposing ends by base pairing. To achieve this, polymerase activity efficiently synthesized DNA across discontinuities in the template strand, and nuclease activity removed a limited number of nucleotides back to regions of microhomology. Processing was suppressed for DNA ends that could be ligated directly, biasing the reaction to preserve DNA sequence and maintain genomic integrity. DNA sequence internal to the ends influenced the spectrum of processing events for noncompatible ends. Furthermore, internal DNA sequence strongly influenced joining efficiency, even in the absence of processing. These results support a model in which DNA‐PKcs plays a central role in regulating the processing of ends for NHEJ.

[1]  T. Paull,et al.  A mechanistic basis for Mre11-directed DNA joining at microhomologies. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  C. Benoist,et al.  Mice lacking TdT: mature animals with an immature lymphocyte repertoire. , 1993, Science.

[3]  S. West,et al.  Involvement of human polynucleotide kinase in double‐strand break repair by non‐homologous end joining , 2002, The EMBO journal.

[4]  H. Pospiech,et al.  The role of DNA polymerase activity in human non-homologous end joining. , 2001, Nucleic acids research.

[5]  R. M. Mason,et al.  The joining of non-complementary DNA double-strand breaks by mammalian extracts. , 1996, Nucleic acids research.

[6]  S. Thode,et al.  A novel pathway of DNA end-to-end joining , 1990, Cell.

[7]  O. Hammarsten,et al.  Activation of DNA-dependent Protein Kinase by Single-stranded DNA Ends* , 2000, The Journal of Biological Chemistry.

[8]  Elke Feldmann,et al.  DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining , 2000, Nucleic Acids Res..

[9]  T. Kunkel,et al.  Implication of DNA Polymerase λ in Alignment-based Gap Filling for Nonhomologous DNA End Joining in Human Nuclear Extracts* , 2004, Journal of Biological Chemistry.

[10]  L. Loeb,et al.  Werner Syndrome Protein , 1998, The Journal of Biological Chemistry.

[11]  M. Fairman,et al.  The identification and characterization of mammalian proteins involved in the rejoining of DNA double-strand breaks in vitro. , 1996, Mutation research.

[12]  J. Turchi,et al.  Differential activation of DNA-PK based on DNA strand orientation and sequence bias , 2005, Nucleic acids research.

[13]  F. Alt,et al.  Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. , 1994, Science.

[14]  K. Sperling,et al.  V(D)J rearrangement in Nijmegen breakage syndrome. , 2000, Molecular immunology.

[15]  L. Loeb,et al.  Werner Syndrome Protein II . CHARACTERIZATION OF THE INTEGRAL 3 9 3 5 9 DNA EXONUCLEASE * , 1998 .

[16]  L. Distel,et al.  Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome. , 2000, Molecular immunology.

[17]  R. Kornberg,et al.  Structure of DNA‐dependent protein kinase: implications for its regulation by DNA , 1999, The EMBO journal.

[18]  F Liang,et al.  Chromosomal double-strand break repair in Ku80-deficient cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Lieber,et al.  Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells , 1997, Nature.

[20]  T. Paull,et al.  Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. , 1999, Genes & development.

[21]  M. Resnick,et al.  Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. , 2000, Mutation research.

[22]  M. Gray,et al.  I. DNA HELICASE AND DNA EXONUCLEASE RESIDE ON THE SAME POLYPEPTIDE , 1998 .

[23]  T. Ried,et al.  Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX. , 2000, Science.

[24]  P. Rouet,et al.  Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. , 1994, Molecular and cellular biology.

[25]  J. J. Espinosa-Aguirre,et al.  Induction of microsomal enzymes in liver of rats treated with cyclohexanol. , 1996, Mutation research.

[26]  T. Kunkel,et al.  The Frameshift Infidelity of Human DNA Polymerase λ , 2003, Journal of Biological Chemistry.

[27]  T. Komori,et al.  Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. , 1993, Science.

[28]  O. Hammarsten,et al.  DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  T. Kunkel,et al.  The frameshift infidelity of human DNA polymerase lambda. Implications for function. , 2003, The Journal of biological chemistry.

[30]  M. Schlissel,et al.  Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination , 1998, Molecular and Cellular Biology.

[31]  Thomas Ried,et al.  Response to RAG-mediated V(D)J cleavage by NBS1 and γ-H2AX , 2000 .

[32]  D. Chan,et al.  The DNA-dependent Protein Kinase Is Inactivated by Autophosphorylation of the Catalytic Subunit (*) , 1996, The Journal of Biological Chemistry.

[33]  Yunmei Ma,et al.  Hairpin Opening and Overhang Processing by an Artemis/DNA-Dependent Protein Kinase Complex in Nonhomologous End Joining and V(D)J Recombination , 2002, Cell.

[34]  J. Hoeijmakers,et al.  The role of DNA dependent protein kinase in synapsis of DNA ends , 2003, Nucleic acids research.

[35]  Yanbin Zhang,et al.  Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase μ , 2001, Molecular and Cellular Biology.

[36]  J. Griffith,et al.  Synapsis of DNA ends by DNA‐dependent protein kinase , 2002, The EMBO journal.

[37]  D. Chan,et al.  Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. , 2001, The Journal of biological chemistry.

[38]  M. Lieber,et al.  Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. , 1994, Science.

[39]  M. Connelly,et al.  DNA-dependent protein kinase catalytic subunit: A relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product , 1995, Cell.

[40]  S. West,et al.  Binding of Inositol Phosphate to DNA-PK and Stimulation of Double-Strand Break Repair , 2000, Cell.

[41]  W. Dynan,et al.  Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein. , 1999, Nucleic acids research.

[42]  A. Tomkinson,et al.  Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. , 2001, Molecular cell.

[43]  A. Fischer,et al.  Artemis, a Novel DNA Double-Strand Break Repair/V(D)J Recombination Protein, Is Mutated in Human Severe Combined Immune Deficiency , 2001, Cell.

[44]  D. Ramsden,et al.  Association of DNA Polymerase μ (pol μ) with Ku and Ligase IV: Role for pol μ in End-Joining Double-Strand Break Repair , 2002, Molecular and Cellular Biology.

[45]  L. Povirk,et al.  Accurate in Vitro End Joining of a DNA Double Strand Break with Partially Cohesive 3′-Overhangs and 3′-Phosphoglycolate Termini , 2001, The Journal of Biological Chemistry.

[46]  C. Farnet,et al.  DNA end-joining in extracts from human cells. , 1995, Biochemical and biophysical research communications.

[47]  F. Alt,et al.  Impairment of V(D)J recombination in double-strand break repair mutants. , 1993, Science.

[48]  P. Baumann,et al.  DNA end-joining catalyzed by human cell-free extracts. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[49]  D. Ramsden,et al.  Ku complex interacts with and stimulates the Werner protein. , 2000, Genes & development.