Molecular characterization of ionizing radiation-hypersensitive mutant M10 cells

Area of Science:

• Molecular Biology
• Genetics
• Radiation Biology

Background:

• Mouse lymphoma L5178Y cell line derivative M10 exhibits sensitivity to ionizing radiation.
• M10 cells show a defect in the rejoining of DNA double-strand breaks (DSBs).
• Complementation tests and chromosome transfer suggested M10 belongs to X-ray cross-complementation (XRCC) group 4.

Purpose of the Study:

• To investigate the genetic basis of ionizing radiation sensitivity in the M10 cell line.
• To identify the specific mutation responsible for the DNA repair defect in M10 cells.
• To confirm the role of the identified gene in DNA double-strand break repair and radiation resistance.

Main Methods:

• Sequence analysis of the Xrcc4 cDNA in M10 cells.
• Analysis of Xrcc4 gene expression from mutant and wild-type alleles.
• Functional rescue of M10 cells by transfecting with murine Xrcc4 cDNA.

Main Results:

• A transversion mutation (A to T at position 370) in the Xrcc4 cDNA was identified, leading to an arginine to termination codon change at position 124.
• The mutation was present in one allele, with gene expression exclusively from the mutant allele.
• Transfection with wild-type murine Xrcc4 cDNA fully restored radioresistance to M10 cells.

Conclusions:

• M10 is a novel Xrcc4-deficient cell line.
• The identified mutation in Xrcc4 is causative for the radiation sensitivity and DSB repair defect in M10 cells.
• This finding reinforces the critical role of XRCC4 in DNA double-strand break repair and maintaining genomic stability.