Gene amplification in human cells knocked down for RAD54.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Gene amplification in mammalian cells is linked to genome instability caused by DNA double-strand breaks (DSBs).
• DSB repair occurs via non-homologous end-joining (NHEJ) and homologous recombination (HR).
• Previous work indicated NHEJ defects elevate gene amplification; this study investigates single or combined DSB repair pathway defects.

Purpose of the Study:

• To investigate the impact of single or combined defects in DSB repair pathways on gene amplification.
• To analyze the role of RAD54 and DNA-PKcs in DNA repair and gene amplification frequency.

Main Methods:

• Human cell lines with constitutive knock-down of RAD54 and/or DNA-PKcs using RNA interference were created.
• Cells were analyzed for radiosensitivity and capacity to generate amplified DNA.
• Cytogenetic analysis of amplicons was performed to understand the mechanisms of copy number increase.

Main Results:

• Both RAD54 and DNA-PKcs deficient cells exhibited hypersensitivity to γ-irradiation.
• These deficient cells generated methotrexate-resistant colonies at a higher frequency than proficient cells.
• Isochromosome formation was identified as a prevalent mechanism for copy number increase in RAD54-deficient cells.

Conclusions:

• DSB repair defects influence the organization of amplified DNA.
• High frequency of isochromosome formation in RAD54-deficient cells suggests HR proteins may prevent centromeric rearrangements.