PARENT SESSION

Genomic Maintenance & Repair

(PP355) The FANCG Fanconi anemia cancer suppressor protein helps minimize spontaneous and radiation-induced chromosomal rearrangements.

Thompson, Larry^*,1, Hinz, John¹, Salazar, Edmund ¹, ¹ Biosciences Directorate, Livermore, CA

ABSTRACT- To study the Fanconi anemia pathway in hamster CHO cells we made an isogenic knockout mutant of fancg and fully corrected this mutant by transfecting the complementing hamster gene on a BAC clone. Fancg mutant cells grow almost normally and have increased sensitivity to killing by both DNA interstrand crosslinking chemicals and methylating agents. Fancg cells have slightly increased sensitivity (1.2 fold) to killing by ionizing radiation (IR), but, interestingly, there is no change in the cell cycle dependence of IR sensitivity. We think this finding is consistent with Fancg acting only during S phase by promoting chromosome stability when replication forks encounter damage. Fancg cells have normal spontaneous chromosomal aberrations and normal IR-induced Rad51 focus formation, indicating proficient homologous recombination (HR). Importantly, spontaneous gene amplification rates for the CAD and DHFR loci are elevated 3- to 4-fold as shown by fluctuation analysis. We find a reduced spontaneous mutation rate at the hprt locus in fancg cells as well as reduced mutagenesis at this locus produced by UV-C, ethylnitrosourea, and -rays delivered to G1-synchronized cells. These results suggest that Fancg is important for replicating DNA containing poorly repaired oxidative base damage produced by IR. Potentially mutagenic base damage in or near the hprt gene may be converted to lethal events in fancg cells. This is the first example of a mammalian cell showing reduced spontaneous or IR-induced mutation rates for the hprt locus. Based on these findings and the complex FA literature, we propose that the main role of most FA proteins, mediated by monoubiquitination and nuclear focus formation of FANCD2, is to: (a) promote mutagenic translesion synthesis past oxidative DNA damage and thereby prevent the collapse of DNA replication forks at the cost of producing point mutations; (b) promote the restoration of stand continuity, by HR, of broken forks that arise spontaneously or from mutagen treatment. We predict that the FA proteins are needed for efficient replication of DNA containing non-double-strand-break bistranded, clustered damage from low-dose IR. (Work done under the auspices of the US DOE by the Univ. of Calif., LLNL, under Contract No. W-7405-Eng-48 and funded by the DOE Low-Dose Program and NCI/NIH grant CA89405.)

Key words: spontaneous mutation rate, gene amplification rate, cell cycle radiosensitivity, chromosomal instability