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

Yanguo Liu, Li Deng, Son C. Nguyen, Catherine L. Au, Changshun Shao, Jay A. Tischfield, Li Liang

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

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 (puror) colonies after culturing reporter cells in medium containing puromycin. The frequency of puror cells can be greatly increased by site-directed DSB inside the insertion. Our results also demonstrated that the frequency of puror 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.

Original languageEnglish (US)
Pages (from-to)140-144
Number of pages5
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume731
Issue number1-2
DOIs
StatePublished - Mar 1 2012

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Keywords

  • Cell-based reporter
  • Double-strand break repair
  • Microhomology-mediated end joining

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