New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant
| Autor: | James B. Wilson, N. Alice Yamada, Nigel J. Jones, Cynthia B. Thomas, John M. Hinz, Irene M. Jones, Larry H. Thompson, Robert S. Tebbs, Edmund P. Salazar |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
DNA Replication
DNA Repair RAD51 Fluorescent Antibody Technique CHO Cells Biology Transfection Biochemistry chemistry.chemical_compound Cricetulus FANCG Fanconi anemia Cricetinae Radiation Ionizing medicine Animals Fanconi Anemia Complementation Group G Protein Molecular Biology Genetics Chinese hamster ovary cell Cell Cycle DNA replication Wild type Cell Biology Methyl Methanesulfonate medicine.disease Molecular biology Methyl methanesulfonate DNA-Binding Proteins Cross-Linking Reagents Fanconi Anemia chemistry Cytoprotection Gene Targeting Mutation Rad51 Recombinase Homologous recombination Mutagens Plasmids |
| Zdroj: | DNA Repair. 4:11-22 |
| ISSN: | 1568-7864 |
| Popis: | The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are largely unknown. By constructing and characterizing a null fancg mutant (KO40) of hamster CHO cells, we show that FancG protects cells against a broad spectrum of genotoxic agents. KO40 is consistently hypersensitive to both alkylating agents that produce monoadducts and those that produce interstrand crosslinks. KO40 cells were no more sensitive to mitomycin C (3×) and diepoxybutane (2×) than to 6-thioguanine (5×), ethylnitrosourea (3×), or methyl methanesulfonate (MMS) (3×). These results contrast with the pattern of selective sensitivity to DNA crosslinking agents seen historically with cell lines from FA patients. The hypersensitivity of KO40 to MMS was not associated with a higher level of initial DNA single-strand breaks; nor was there a defect in removing MNU-induced methyl groups from DNA. Both control and MMS-treated synchronized G1-phase KO40 cells progressed through S phase at a normal rate but showed a lengthening of G2 phase compared with wild type. MMS-treated and untreated early S-phase KO40 cells had increased levels of Rad51 foci compared with wild type. Asynchronous KO40 treated with ionizing radiation (IR) exhibited a normal Rad51 focus response, consistent with KO40 having only slight sensitivity to killing by IR. The plating efficiency and doubling time of KO40 cells were nearly normal, and they showed no increase in spontaneous chromosomal aberrations or sister chromatid exchanges. Collectively, our results do not support a role for FancG during DNA replication that deals specifically with processing DNA crosslinks. Nor do they suggest that the main function of the FA protein “pathway” is to promote efficient homologous recombination. We propose that the primary function of FA proteins is to maintain chromosomal continuity by stabilizing replication forks that encounter nicks, gaps, or replication-blocking lesions. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect