NBN phosphorylation regulates the accumulation of MRN and ATM at sites of DNA double-strand breaks

Abstract

In response to ionizing radiation, the MRE11/RAD50/NBN complex re-distributes to the sites of DNA double-strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for the activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine-to-alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine-to-glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared with control cells, whereas blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.

Figure 1

I-PpoI protein purification and transduction into human cells. (A) I-PpoI protein purified from E. coli was detected by Coomassie staining. (B) pcDNA3.1 containing a synthetic I-PpoI site was linearized by PvuI digestion, and incubated with either control (Con) protein (produced from pET45b(+) empty vector transformed cells) or purified I-PpoI protein at 37°C overnight. Products were separated on agarose gel. (C) ILB1, ILB1-[NBN] or ILB1-[NBN-S>A] cells were transduced with purified I-PpoI protein complexed with cell penetrating peptides (CPP). Cells were collected at 0, 1, 2, 3 and 5 hours after transduction. Total protein lysate was separated on a 4–12% gradient Bis-Tris gel, transferred to Immobilon-P transfer membrane and immunoblotted with a monoclonal anti-His antibody and a monoclonal anti-tubulin antibody respectively. (D) ILB1-[NBN] cells were transduced with purified I-PpoI protein complexed with CPP. Cells were collected at 0, 1, 3 and 5 hours after transduction. Cytoplasmic and nuclear fractions were prepared and separated on a 4–12% gradient Bis-Tris gel, transferred to Immobilon-P transfer membrane and immunoblotted with anti-His antibody, anti-Hsp90 (as a cytoplasmic control) and anti-p84 (as a nuclear control).

Figure 2

Transduced I-PpoI induces site-specific DNA cleavage in vivo. (A) In ILB1-[NBN] cells, the yield of PCR products spanning each of 10 identified I-PpoI sites at different time points was compared with that from a reference sequence on chromosome 1 that did not contain an I-PpoI site. Data were averaged from three independent experiments, and the error bars represent mean ± SD. (B) In ILB1, ILB1-[NBN], ILB1-[NBN-S>A], ILB1-[NBN-S>E] and 411BRneo cells, yield of PCR product at a single I-PpoI site in the DAB1 gene on chromosome 1 relative to a reference sequence lacking an I-PpoI site on the same chromosome. The data represent the mean ± SD from independent triplicate experiments each including two technical replicates. (C) In BLCL-309 and HEK293T cells, DNA and protein samples were collected at 0, 1 and 3 hours post I-PpoI transduction. Yield of PCR product at a single I-PpoI site in the DAB1 gene on chromosome 1 was compared with that from a reference sequence on chromosome 1 that did not contain an I-PpoI site (lower panel). Western blots show effective and rapid protein transduction (upper panel).

Figure 3

I-PpoI protein transduction activates ATM-dependent DNA damage responses. (A) In the upper panel, cells treated with I-PpoI for the indicated amount of time were lysed, nuclear extracts were prepared and separated by 3–8% gradient gel, transferred to Immobilon-P transfer membranes, and immunoblotted with antibodies against ATM-phosphor-serine1981 (ATM-pS1981) and total ATM. In the lower panel, lysates from cells treated with I-PpoI for the indicated times were immunoprecipitated with an anti-NBN antibody and immunoblotted with antibodies against NBN-phospho-serine343 (NBN-pS343) and total NBN. (B) ILB1-[NBN] cells were transduced with PBS (N/A), either control protein (Con) or purified I-PpoI protein (I-PpoI) using a protein delivery reagent (Chariot) and collected at 1 and 2 hours after transduction. ChIP assays were performed using either no antibody, or antibody against γH2AX. The immunoprecipitated DNA was quantitated by PCR using a primer set adjacent to the I-PpoI site in the DAB1 gene on chromosome 1 or primer set targeting a housekeeping gene, GAPDH. (C) ILB1-[NBN] cells were mock treated (No treatment), treated with CPP alone (CPP), or transduced with either control protein (Con-treated) or purified I-PpoI protein (I-PpoI-treated) complexed with CPP. Cells were fixed at 0, 1, 2, 4 and 6 hours after transduction and stained with γH2AX and DAPI. Low (20X) and high (40X) magnification images are shown for each time point.

Figure 4

Mutation of ATM phosphorylation sites on NBN alters the timing of ATM and NBN recruitment to the sites of damage. (A-B) ILB1, ILB1-[NBN], ILB1-[NBN-S>A] and ILB1-[NBN-S>E] cells were transduced with purified I-PpoI protein using CPP. Cells were lysed at 0, 1, 3, 5 and 8 hours after transduction. ChIP assays were performed using antibodies against NBN (A), and ATM (B).

Figure 4

Mutation of ATM phosphorylation sites on NBN alters the timing of ATM and NBN recruitment to the sites of damage. (A-B) ILB1, ILB1-[NBN], ILB1-[NBN-S>A] and ILB1-[NBN-S>E] cells were transduced with purified I-PpoI protein using CPP. Cells were lysed at 0, 1, 3, 5 and 8 hours after transduction. ChIP assays were performed using antibodies against NBN (A), and ATM (B).

Figure 5

Mutation of ATM phosphorylation sites on NBN does not alter the phosphorylation of ATM and its downstream effectors. (A) ILB1-[NBN], ILB1-[NBN-S>A] and ILB1-[NBN-S>E] cells were transduced with purified I-PpoI protein and lysed at 0, 1, 3 and 8 hours after transduction. Nuclear extracts was prepared. Equivalent amounts of nuclear extract from the same preparation were separated on a 3–8% Tris-Acetate gel and a 4–12% Bis-Tris gel individually, transferred to Immobilon-P transfer membranes, and immunoblotted with antibodies against ATM-phosphor-serine1981 (ATM-pS1981, on Tris-Acetate gel), ATM (on Bis-Tris gel), (B) CHK2-phosphor-Tyrosine68 (CHK pT68, on Tris-Acetate gel) and CHK2 (on Bis-Tris gel), (C) SMC1-phosphor-Serine957 (SMC1 S957, on Bis-Tris gel) and SMC1 (on Tris-Acetate gel).