Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control

Abstract

The role of protein arginine methylation in the DNA damage checkpoint response and DNA repair is largely unknown. Herein we show that the MRE11 checkpoint protein is arginine methylated by PRMT1. Mutation of the arginines within MRE11 severely impaired the exonuclease activity of MRE11 but did not influence its ability to form complexes with RAD50 and NBS1. Cells containing hypomethylated MRE11 displayed intra-S-phase DNA damage checkpoint defects that were significantly rescued with the MRE11-RAD50-NBS1 complex. Our results suggest that arginine methylation regulates the activity of MRE11-RAD50-NBS1 complex during the intra-S-phase DNA damage checkpoint response.

Figure 1.

MRE11 contains asymmetrically dimethylated arginines. (A) Antibodies recognizing sDMA (SYM10) or aDMA (ASYM25) were used to perform immunoprecipitations from HeLa cell extracts and, after washing, bound proteins were analyzed by Western blotting to detect MRE11. The total cell lysate (TCL) shows 10% of the extract used in each binding assay. The migration of MRE11 is indicated on the right. The molecular mass markers are shown on the left in kilodaltons. Normal rabbit serum (NRS) was used as a negative control. (B) MRE11 was immunoprecipitated from HeLa cell extracts and, after washing, bound proteins were analyzed by immunoblotting using SYM10 and ASYM25. Many methylated proteins are visualized in the TCL, and the migration of MRE11 is shown with an arrow.

Figure 2.

Arginine methylation of MRE11 by PRMT1. (A) Recombinant GST-PRMTs or PRMT5 immunoprecipitated from HeLa cells were incubated with GST-MRE11 (554-680) in the presence of [³H]-AdoMet as the methyl donor. Proteins were separated by SDS-PAGE and visualized by Coomassie staining (left) and the [³H]-labeled proteins visualized by fluorography (right). (B) PRMT1^+/+ and PRMT1^-/- were metabolically labeled with [methyl-³H]-L-methionine, and cell lysates were immunoprecipitated with Sam68, Sm (Ana128; Cappel), or MRE11 antibodies. The in vivo methylated proteins were visualized by fluorography after SDS-PAGE. (C) Equivalent amount of TCL from normal ES cells or ES cells PRMT1^-/- was immunoblotted with anti-MRE11 antibodies.

Figure 3.

The MRE11 GAR domain regulates the exonuclease activity. (A) HIS-tagged human MRE11 wild-type, R/A, and R/K baculoviruses were generated, and the corresponding proteins were purified to homogeneity from insect cells. Purified MRE11, MRE11R/A, and MRE11R/K were visualized by Coomassie blue (top panel) and immunoblotted with MRE11 (middle panel) and ASYM25 (bottom panel). (B) To determine whether the methylated arginines are necessary for the formation of the MRN complex, cells infected with NBS1, RAD50, and either MRE11, MRE11R/A, or MRE11R/K baculoviruses were lysed and immunoprecipitated with beads alone or anti-NBS1 antibodies. The presence of RAD50 and MRE11 as well as NBS1 was visualized by immunoblotting. Their identities are indicated on the right. (C) Nuclease assays were performed with the indicated amount of each of MRE11 WT, MRE11R/A, or MRE11R/K, in 5 mM MnCl₂ on a [³²P]-5′-labeled at one end 63-bp dsDNA substrate. Reactions were incubated for 60 min at 37°C before separation on a 10% denaturing polyacrylamide gel and visualized by autoradiography.

Figure 4.

Arginine methylation of MRE11 regulates the intra-S-phase checkpoint. (A) DNA synthesis following DNA damage in HeLa cells pretreated with the methylase inhibitor MTA, in cells treated with PRMT1-siRNA, or in A-TLD cells. DNA synthesis was assessed 1 h after 50 μM etoposide treatment. The results represent an average of six independent experiments. (B) Cell extracts from mock- and PRMT1-siRNA-transfected HeLa cells were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with PRMT1, Sam68, Arg587, and MRE11 antibodies as indicated. (C) DNA synthesis following DNA damage in PRMT1-siRNA cells protein transfected with 100 ng of the MRE11 complex (MRN) or MRE11 R/A alone using Chariot. DNA synthesis was assessed 1 h after 50 μM etoposide treatment. The results represent an average of six independent experiments. The asterisk represents a significant difference between PRMT1 RNAi and PRMT1 RNAi + MRN (P < 0.01). (D) S-phase progression was followed using flow cytometry of PRMT1^+/+ ES cells (top panels) or PRMT1^-/- ES cells (bottom panels) 16 h following either mock treatment (left panels) or DNA damage induced with a low dose of 500 nM etoposide (right panels). The percentage of cells in the G₀/G₁, S, or G₂/M phases of the cell cycle is indicated.