33. Nonsense Mutation at 124 in the XRCC4 of Radiosensitive Cell Line M10

Masahiko Mori, Hiromi Itsukaichn and Koki Sato* (*Kinki Univ.)

Keywords: DNA-double strand breaks repair, XRCC4 gene, potnt mutatton

The XRCC4 gene is required for the repair of DNA double-strand breaks in mammalian cells. Without XRCC4, cells are hypersensutuve to IOIIIZIIIB radiation and deficient for V(D)J recombination. The mouse lymphoma L5178Y cell mutant MID is defective in DNA-double strand breaks repair and hypersensitive to ionizing radiation. The complemen tation test by expression of human XRCC4 cDNA in MID cells suggested that the MID cell line belongs to a x-ray cross complementing group 4 (XRCC4)

We isolated the open reading frame (ORF) of XRCC4 cDNA from MID and 1,5178Y cells and compared them at DNA sequence level. Consequently point mutation A to T (370) was discovered in XRCC4 cDNA of MID cells. l'he mutation is located in exon 4 and results in an arginine (124) to termination codon. To verify our findings, we assessed the mutation with genomic DNA prepared from L5178Y and MID cells, since substitution oc eurring in MID created the restriction enzyme Nde I recognition site. Interestingly, analysis of genomic DNA revealed the presence of two kinds of chromosomes related to the mutated region in MID cells. One allele had the Nde I recognition site while the other did not. This means that the mutation took place in only one of two alleies. Indeed, only T peak appeared at nucleotide number 370 in the cDNA sequence pattern, while A and T peaks clearly appeared at the corresponding nucleotide in the genomic DNA sequence pattern. To analyze complementation of the radiosensitivity characteristic of MID cells the pME18S puromycin vector alone or pME18S murine XRCC4 cDNA was introduced into MID cells and stable transfectants were selected by use of the puromycin resistant marker. The clones were corrected for radiosensitivity similar to that of the wild type, L5178Y, levels. The clones formed transfections with pVIE18S vector alone wihich showed the radiosensitivity of MID cells.

Fig.13. Nonsense mutation in murine XRCC4 gene in M10 cells