Exonuclease Activity is Essential for Mre11 in Controlling Microhomology Mediated End Joining of DNA Double-Strand Breaks

Abstract

Mre11, in a complex with Rad50 and NBS1, is critical in DNA damage response. Patients with germline mutations in Mre11 share similar clinical presentations as Ataxia-Telangiectasia (AT) patients. Loss of Mre11 results in increased radiosensitivity and chromosomal instability. Previously we have shown that Mre11 may play a direct role in DNA double-strand break (DSB) repair by promoting one of the major nonhomologous end-joining (NHEJ) subpathways, microhomology mediated end joining (MMEJ). The objective of this study is to elucidate the mechanism underlying Mre11's promotion of MMEJ by defining the functional domains of Mre11 involved in this process. 1) A NHEJ reporter susbstrate with two I-SceI restriction endonuclease sites was integrated into the chromosome of human embryonic kidney (HEK) 293 cells, and DNA DSBs were generated by transient transfection of an I-SceI expression vector. 2) Mre11 was knocked down using siRNA, and MMEJ was quantified by real-time PCR using primers and probes specific for MMEJ repair products. These products were also confirmed by direct sequencing at the junction site. 3) To demonstrate that the Mre11 siRNA phenotype is not due to off target effects, a siRNA-resistant Mre11 cDNA was used to rescue the MMEJ phenotype. 4) To understand the mechanism of Mre11 in MMEJ, functionally deficient siRNA resistant Mre11 mutants were used in siRNA rescue experiments. 5) Cell cycle distribution and viability, which influence repair processes and also are affected by Mre11, were measured. 6) To determine the effect of different Mre11 status on cytotoxic response to radiation, clonogenic assays were performed. 1) Mre11 knockdown resulted in a 7.4 fold decrease (p < 0.01) in MMEJ DSB repair efficiency and a 1.7 fold decrease (p < 0.01) in the surviving fraction after radiation. 2) SiRNA-resistant wildtype Mre11 can fully rescue this MMEJ deficiency. 3) However, the MMEJ deficiency cannot be rescued by Mre11-3, a phosphodiesterase mutant, nor Mre11-R/A, a methylation resistant mutant. Interestingly, while both mutants maintain the ability to form a complex with Rad50 and Nbs1 and bind DNA, the exonuclease activity of both mutants is abrogated. 4) Mre11 status does not affect cell cycle distribution or cell viability in this study. This study establishes that Mre11 controls MMEJ-mediated DSB repair in human cells. The repair function of Mre11 may be one of the underlying mechanisms for the enhanced radiosensitivity seen in Mre11 deficient cells in this study and for the hypersensitivity to radiation in patients with germline Mre11 mutations. Furthermore, we have demonstrated that the exonuclease activity of Mre11 is essential for its function in MMEJ. Inhibition of Mre11 exonuclease activity can be a beneficial therapeutic target to sensitize cancer cells to radiotherapy.