title: A human cell-based reporter detects microhomology-mediated end joining.
abstract: DNA double-strand breaks (DSBs) are most often repaired by two pathways in mammalian cells, homologous recombination or non-homologous end joining. Biochemical and genetic studies showed that DSBs can also be joined via microhomology-mediated end joining (MHEJ), which is always mutagenic and may result in diseases, such as cancer. In this study we established a human cell-based reporter system to determine the prevalence of MHEJ events and factors that modulate MHEJ. A nonfunctional puromycin acetyltransferase (Pac) gene, disrupted by an insertion flanked by two microhomologous repeats, was integrated into chromosomes of human HT1080 cells. Repair of DSBs via MHEJ using the repeats resulted in deletion of the insertion and restoration of the Pac gene function, thus rendering the cells puromycin resistant. Our results showed that MHEJ spontaneously occurs at the reporter locus (loci), manifested by formation of puromycin resistant (puro(r)) colonies after culturing reporter cells in medium containing puromycin. The frequency of puro(r) cells can be greatly increased by site-directed DSB inside the insertion. Our results also demonstrated that the frequency of puro(r) cells is affected by the length of the repeat and by the size of the intervening sequence. Thus, this cell-based assay provides a platform for evaluating factors modulating in vivo MHEJ.
authors.affiliation: Institute of Basic Medical Sciences, Shandong University, Jinan, China.
authors.forename: Yanguo
authors.initials: Y
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Deng
authors.forename: Son C
authors.initials: SC
authors.lastname: Nguyen
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Au
authors.forename: Changshun
authors.initials: C
authors.lastname: Shao
authors.forename: Jay A
authors.initials: JA
authors.lastname: Tischfield
authors.forename: Li
authors.initials: L
authors.lastname: Liang
mesh_terms: D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D019008:Drug Resistance, Neoplasm; D005821:Genetic Techniques; D006801:Humans; D011691:Puromycin / Q000494:pharmacology
chemical_list: D011691:Puromycin