Mechanism of suppression of chromosomal instability by DNA polymerase POLQ
| Autor: | Sylvie Doublié, David W. Wyatt, Sean C. Hensley, Richard D. Wood, Kevin M. McBride, Göran O. Bylund, Junya Tomida, Yunxiang Mu, Erik Johansson, Kei Ichi Takata, Matthew J. Yousefzadeh, Dale A. Ramsden |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Genome instability
Cancer Research DNA End-Joining Repair DNA polymerase DNA Polymerase Theta Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology) Molecular Biology Microbiology Biochemistry or Biopharmacy) DNA-Directed DNA Polymerase QH426-470 Biochemistry 0302 clinical medicine Annan medicinsk grundvetenskap Genetics of the Immune System DNA Breaks Double-Stranded Other Basic Medicine Genetics (clinical) Cells Cultured Genetics 0303 health sciences Ku70 B-Lymphocytes biology 030220 oncology & carcinogenesis Class Switching Female Medical Genetics Metabolic Networks and Pathways Research Article DNA repair Base pair DNA damage Immunology Bone Marrow Cells DNA replication Non-Homologous End Joining 03 medical and health sciences Bleomycin Chromosomal Instability Cancer Genetics Animals Humans Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi) molekylärbiologi mikrobiologi biokemi eller biofarmaci) Molecular Biology Ecology Evolution Behavior and Systematics 030304 developmental biology Medicinsk genetik Biology and life sciences DNA synthesis Radiobiology DNA Cell Biology Immunoglobulin Class Switching Mice Mutant Strains Mice Inbred C57BL HEK293 Cells biology.protein Clinical Immunology |
| Zdroj: | PLoS Genetics PLoS Genetics, Vol 10, Iss 10, p e1004654 (2014) |
| ISSN: | 1553-7404 |
| Popis: | Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone. Author Summary The reason for the hypersensitivity of POLQ-defective mammalian cells to ionizing radiation has been elusive. Here we show that POLQ-defective mammalian cells are selectively susceptible to double-strand breaks in DNA. We present experiments in mammalian cells showing that a specific double-strand break repair pathway is POLQ-dependent. To analyze the repair function in more detail, we examined class switch joining between DNA segments in antibody genes. Insertions of DNA bases are sometimes found at the joins between such segments, but the origin of these insertions has been mysterious. We show that this class of insertion joins during immunoglobulin class-switching is entirely POLQ-dependent. In experiments with purified human POLQ protein, we found a novel biochemical mechanism explaining the formation of the insertions. POLQ has a unique biochemical ability to extend DNA with minimal base pairing. Finally, we examined the biological consequences for chromosome stability. Unexpectedly, the Burkitt lymphoma translocation (a major cancer-associated genome instability) is enhanced in the absence of POLQ. This alters the current view about the action of DNA end joining in mammalian cells, revealing that a POLQ-dependent DNA repair pathway combats potentially damaging chromosome translocations. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|