A human cell-based reporter detects microhomology-mediated end joining.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions repaired by homologous recombination or non-homologous end joining.
• Microhomology-mediated end joining (MHEJ) is an alternative, mutagenic DSB repair pathway implicated in diseases like cancer.

Purpose of the Study:

• To establish a human cell-based reporter system for quantifying MHEJ prevalence.
• To investigate factors that modulate MHEJ activity in vivo.

Main Methods:

• A reporter system was created in HT1080 cells using a disrupted puromycin acetyltransferase (Pac) gene flanked by microhomologous repeats.
• MHEJ repair of DSBs restored Pac gene function, enabling selection of puromycin-resistant colonies.
• The impact of repeat length and intervening sequence size on MHEJ frequency was assessed.

Main Results:

• Spontaneous MHEJ events were detected at the reporter locus, indicated by puromycin-resistant colony formation.
• Site-directed DSBs within the insertion significantly increased MHEJ frequency.
• MHEJ frequency was demonstrably influenced by the length of microhomologous repeats and the size of the deleted sequence.

Conclusions:

• The developed cell-based assay effectively quantifies MHEJ events in human cells.
• This system provides a platform for evaluating modulators of in vivo MHEJ.
• Understanding MHEJ is crucial due to its mutagenic nature and association with diseases.