Distinct Mechanisms of Nonhomologous End Joining in the Repair of Site-Directed Chromosomal Breaks with Noncomplementary and Complementary Ends

Abstract

DNA double-strand breaks (DSBs) are considered the most important type of DNA damage inflicted by ionizing radiation. The molecular mechanisms of DSB repair by nonhomologous end joining (NHEJ) have not been well studied in live mammalian cells, due in part to the lack of suitable chromosomal repair assays. We previously introduced a novel plasmid-based assay to monitor NHEJ of site-directed chromosomal I-SceI breaks. In the current study, we expanded the analysis of chromosomal NHEJ products in murine fibroblasts to focus on the error-prone rejoining of DSBs with noncomplementary ends, which may serve as a model for radiation damage repair. We found that noncomplementary ends were efficiently repaired using microhomologies of 1-2 nucleotides (nt) present in the single-stranded overhangs, thereby keeping repair-associated end degradation to a minimum (2-3 nt). Microhomology-mediated end joining was disrupted by Wortmannin, a known inhibitor of DNA-PKcs. However, Wortmannin did not significantly impair the proficiency of end joining. In contrast to noncomplementary ends, the rejoining of cohesive ends showed only a minor dependence on microhomologies but produced fivefold larger deletions than the repair of noncomplementary ends. Together, these data suggest the presence of several distinct NHEJ mechanisms in live cells, which are characterized by the degree of sequence deletion and microhomology use. Our NHEJ assay should prove a useful system to further elucidate the genetic determinants and molecular mechanisms of site-directed DSBs in living cells.

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

“…We stably integrated a single copy of the NHEJ substrate pHW1 in which the two I-SceI sites are separated by 30 base pairs of intervening sequence (29,30) into the genome of HEK293 cells. A pair of primers flanking the two I-SceI sites and a probe complementary to the sequence resulting from precise end joining was designed for quantitative real-time PCR (Fig.…”

“…Strategy for NHEJ Assays-Methods for analysis of precise NHEJ have been reported previously (29). Forty-eight hours after I-SceI expression, genomic DNA was extracted with Qiagen's DNeasy kit for real-time PCR analysis.…”

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

“…A substrate with a closer I-SceI site has also been described (39). We have shown that the I-SceI-excised fragment can be inverted or translocated and captured at a third DSB (1).…”

Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

“…To explore further the mechanisms that regulate NHEJ subpathways, we have used an assay to create site-specific DSBs within the chromosomally integrated NHEJ substrate pPHW1 in HEK293 cells (32,33), which produces fully complementary cohesive ends. We showed that both precise C-NHEJ and imprecise D-NHEJ pathways are utilized to repair the DSBs in this setting, consistent with a previous observation (33). When the DNA damage response protein Mre11 was silenced, there was an ϳ10 -12-fold reduction in small sequence deletions at the break junction compared with the levels of D-NHEJ in siRNA control cells (Figs.…”

“…The structure of the NHEJ substrate and the strategy to measure NHEJ were described previously (9,32,33) and are depicted in Rejoining of the double-stranded ends by either precise C-NHEJ or error-prone D-NHEJ will reconstitute translation of the gpt ORF, permitting cell survival under XHATM selection and detection of recombinants as colonies grown in XHATM selection medium. Alternatively, error-prone D-NHEJ at either of the single I-SceI sites can also disrupt ATG ART , allowing again for selection of recombinants with XHATM.…”