Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair

Abstract

Mammalian DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol mu protein increase. pol mu also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of gammaH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol mu is thus part of the cellular response to DNA double-strand breaks. pol mu also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol mu in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase beta does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol mu in facilitating joining mediated by these factors. Our data thus support an important role for pol mu in the end-joining pathway for repair of double-strand breaks.

FIG. 1.

Effects of DNA-damaging agents on pol μ expression. HEK cells were exposed to either 12 Gy of IR, 7 J of UV irradiation/m², or 50 μg of mitomycin C (MitC)/ml. Whole-cell extracts were prepared from cells that were left untreated (0) or treated for the indicated times. The blot was probed with anti-pol μ, anti-phosphoserine 15-p53 (S15P-p53), and anti-Ku 80 antibodies.

FIG. 2.

Cellular localization of pol μ. (A) Levels of the pol μ-EGFP fusion protein and those of endogenous pol μ were compared by Western blot analysis, using the pol μ-specific antibody, of 100 μg of extract from HEK cells transfected either with the vector alone (lane 2) or with pol μ-EGFP (lane 3). Fifty nanograms of recombinant pol μ is shown as a marker (lane 1); note that recombinant pol μ migrates slightly more slowly than endogenous pol μ due to the hexahistidine tag. (B) The cellular distribution of the pol μ-EGFP fusion protein was determined by comparing GFP fluorescence to 4′,6′-diamidino-2-phenylindole (DAPI) staining (used to delineate nuclei) in cells transfected with pol μ-EGFP. (C) Cells transfected with pol μ-EGFP were assessed for GFP fluorescence (pol μ-EGFP) without treatment (Untreated), 2 h after treatment with IR (Irradiated), or 8 h after treatment with etoposide (Etoposide). Also shown are untreated and irradiated cells stained in parallel with antibodies to γH2AX (stained red by a rhodamine-conjugated secondary antibody), as well as merged images of pol μ-EGFP and γH2AX fluorescence (Merge).

FIG. 3.

Association of pol μ with Ku. Protein complexes were immunoprecipitated (IP) from whole-cell extracts using mouse IgG (mIgG) or monoclonal antibodies (Abs) against Ku or EGFP. (A) Proteins immunoprecipitated from extracts (xt) from pol μ-EGFP-transfected HEK cells were characterized by Western blot analysis with anti-Rad50 (upper panel), anti-Ku 80 (middle panel) or anti-EGFP (lower panel) antibodies. When present, ethidium bromide (EtBr) was added to a concentration of 100 μg/ml. (B) Immunoprecipitated proteins from Ramos cell extracts were characterized by Western blot analysis with anti-pol μ antibodies (upper panel) or anti-Ku 80 antibodies (lower panel).

FIG. 4.

Protein-DNA complexes involving pol μ, Ku, and X4-LIV. (A) Coomassie blue-stained SDS-PAGE gel of purified recombinant proteins. Lane M, marker; lane 1, 4 μg of X4-LIV; lane 2, 4 μg of Ku heterodimer; lane 3, 2 μg of pol μ; lane 4, 2 μg of TdT; lane 5, 2 μg of pol β. (B) All reactions contained 90 nM ³²P-labeled 60-bp DNA duplex. F, free DNA probe; I, II, and III, species I, II, and III. Ku (5 nM) and X4-LIV (25 nM) were included as indicated (+). pol μ was added at a concentration of 100 nM (lanes 2, 3, 4, and 6), 50 nM (lane 7), or 25 nM (lane 8).

FIG. 5.

Participation of different pol X family members in complexes with end-joining factors. All reactions contained 90 nM ³²P-labeled 60-bp DNA duplex. F, free DNA probe; I, II, III, and IV, species I, II, III, and IV. (A) pol β (100 nM), pol μ (100 nM), Ku (5 nM), and X4-LIV (25 nM) were included as indicated. (B) Ku (5 nM), X4-LIV (10 nM), TdT (25 nM), and α-TdT (0.2 μl) were added as indicated (+). pol μ or pol β was added at 25 nM (+) or 75 nM (+++).

FIG. 6.

Association of pol μ with X4-LIV. (A) All reactions contained 90 nM ³²P-labeled 60-bp DNA duplex and 5 nM Ku. F, free DNA probe; I, II, and III, species I, II, and III. pol μ (100 nM) was included as indicated (+). X4-LIV was added to a concentration of 25 nM (lanes 5 and 6), 10 nM (lanes 4 and 7), 5 nM (lanes 3 and 8), or 2.5 nM (lanes 2 and 9). (B) pol μ and pol β (each at 250 nM) were present in all immunoprecipitations. X4-LIV (50 nM), Ku (100 nM), and sheared calf thymus DNA (∼1 μM) were added as indicated (+). αX4, 1 μl of polyclonal anti-XRCC4 antisera. Input pols, a fraction of reaction input. Immunoprecipitated proteins were detected by Western blot analysis using an anti-pol β antibody (top panel) or an anti-histidine tag antibody (bottom two panels; hexahistidine tags are present on both ligase IV and pol μ). IgH, heavy chain of immunoprecipitating antibody (detected by a species cross-reaction of the anti-mouse Ig secondary antibody used for Western blot analysis).

FIG. 7.

Relative activities of polymerases in gap and break repair. (A) Oligonucleotide sequences and substrate assembly as described in Materials and Methods. Stars indicate locations of ³²P label. (B) ³²P-labeled substrates at 5 nM (see panel A) were present in all reactions. pol μ (25 nM) or pol β (25 nM), Ku (5 nM), X4-LIV (50 nM), and salmon sperm DNA (1.5 μM) were added as noted. The reaction time was 1 min.