Abstract
DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine-arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.
Highlights
DNA double-stranded breaks (DSBs)3 can be produced in physiological and genotoxic processes
Upon sequencing the repair junctions, we found that Mre11 siRNA knockdown suppressed D-nonhomologous end joining (NHEJ) by ϳ10-fold, reflected by a reduction of small deletions in the repair junction, but it had no effect on the efficiency of conservative NHEJ (C-NHEJ)
Strategy and Generation of the human embryonic kidney 293 (HEK293)/pPHW1 Cell Line— To investigate the mechanisms that control the accuracy of NHEJ-mediated DNA DSB repair in mammalian cells, we generated a cell line in which a single copy of the NHEJ substrate pPHW1 was stably integrated into the genome (HEK293/ pPHW1 cells)
Summary
HEK293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin/streptomycin, and glutamine. Southern blotting was performed to confirm that a single copy of pPHW1 was integrated at a single locus. This cell line (HEK293/pPHW1) was maintained in complete Dulbecco’s modified Eagle’s medium containing 2 g/ml puromycin. To create DSB within the chromosomally integrated NHEJ substrate, meganuclease I-SceI was expressed in the HEK293/ pPHW1 cells by transient transfection of the expression plasmid pCMV-I-SceI. The Mre11-R/A protein has a R587A mutation in its GAR region [23, 25] Both mutant proteins lack exonuclease activity and showed resistance to Mre siRNA silencing. The target for Mre siRNA oligonucleotides is located in the 5Ј-untranslated region of Mre mRNA, so the HEK293/pPHW1 cell line containing Mre cDNA (pcDNA3-Myc-Mre11-WT, Mre11R/A, or Mre11-3) can be resistant to Mre siRNA silencing. Proteins were visualized with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:5000; Santa Cruz, CA) followed by the use of an ECL chemiluminescence system (Amersham Biosciences)
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