New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant

Robert S. Tebbs, John M. Hinz, N. Alice Yamada, James B. Wilson, Edmund P. Salazar, Cynthia B. Thomas, Irene M. Jones, Nigel J. Jones, Larry H. Thompson

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)11-22
Number of pages12
JournalDNA Repair
Volume4
Issue number1
DOIs
StatePublished - Jan 2 2005
Externally publishedYes

Keywords

  • FANCG
  • Fanconi anemia
  • Homologous recombination
  • Rad51 foci
  • Replication fork

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Tebbs, R. S., Hinz, J. M., Yamada, N. A., Wilson, J. B., Salazar, E. P., Thomas, C. B., Jones, I. M., Jones, N. J., & Thompson, L. H. (2005). New insights into the Fanconi anemia pathway from an isogenic FancG hamster CHO mutant. DNA Repair, 4(1), 11-22. https://doi.org/10.1016/j.dnarep.2004.06.013