Stimulation of NEIL2-mediated oxidized base excision repair via YB-1 interaction during oxidative stress

Abstract

The recently characterized enzyme NEIL2 (Nei-like-2), one of the four oxidized base-specific DNA glycosylases (OGG1, NTH1, NEIL1, and NEIL2) in mammalian cells, has poor base excision activity from duplex DNA. To test the possibility that one or more proteins modulate its activity in vivo, we performed mass spectrometric analysis of the NEIL2 immunocomplex and identified Y box-binding (YB-1) protein as a stably interacting partner of NEIL2. We show here that YB-1 not only interacts physically with NEIL2, but it also cooperates functionally by stimulating its base excision activity by 7-fold. Moreover, YB-1 interacts with the other NEIL2-associated BER proteins, namely, DNA ligase III alpha and DNA polymerase beta and thus could form a large multiprotein complex. YB-1, normally present in the cytoplasm, translocates to the nucleus during UVA-induced oxidative stress, concomitant with its increased association with and activation of NEIL2. NEIL2-initiated base excision activity is significantly reduced in YB-1-depleted cells. YB-1 thus appears to have a novel regulatory role in NEIL2-mediated repair under oxidative stress.

FIGURE 1. Identification of YB-1 as NEIL2-associated protein

A, SDS-PAGE of the 500 mm salt eluate of NEIL2-FLAG IC (lane 2) or empty vector (lane 1) isolated from HCT116N cell lysates. *, mass spectrometric analysis of the marked band. B, NEIL2-FLAG immunocomplex was tested for the presence of YB-1 by Western analysis (lane 3); lane 2, vector IP as control; lane 1, purified YB-1 as marker; lanes 4 and 5, corresponding cell lysates as input. Lower panel, Western analysis of IC and cell extracts used in the upper panel with anti-FLAG antibody. C, Far Western analysis showing direct interaction between YB-1 (lanes 5, 7, and 9) with WT, and the N-terminal (NTD) and C-terminal domains (CTD) of NEIL2. Lanes 1–3, Coomassie stain of marker, bovine serum albumin (BSA), and YB-1, respectively.

FIGURE 2. Identification of the NEIL2 interacting region of YB-1

A and C, schematic representation of the GST-YB1 deletion mutants (Δ1–Δ5) and (Δ6–Δ9). B and D, Far Western analysis. Various GST-YB-1 deletion mutants were used for the interaction studies with NEIL2 (right panel). Left panel, Coomassie staining of the YB-1 deletion mutants. E, GST pull-down assay. NEIL2 incubated with glutathione-agarose affinity beads containing various deletion mutants of GST-tagged YB-1 (as indicated Δ6–Δ9), blotted with anti-NEIL2 antibody.

FIGURE 3. Stimulation of NEIL2 activity by YB-1

A, NEIL2 (50 fmol) was incubated for 10 min with the 5-OHU-containing ³²P-labeled bubble substrate in the presence of increasing amounts of YB-1 (50 – 400 fmol), and the reaction products were analyzed in a 20% urea-polyacrylamide gel, and the cleaved products were quantitated (lower panel). B, NEIL2 incision activity on 5-OHU-containing ³²P-labeled duplex substrate (lanes 1–5) or 8-oxoG containing ³²P-labeled bubble substrate (lanes 6 –11) in the presence (+) or absence (−) of YB-1. Cleaved products were quantitated as shown in the lower panel. C, NEIL2 incision activity on 5-OHU-containing ³²P-labeled bubble substrate in the presence of different GST-YB1 deletions as indicated in Fig. 2. Lower panel, quantification of the cleaved products. S, substrate; P, product. BSA, bovine serum albumin.

FIGURE 4. Interaction of YB-1 with other NEIL2-associated proteins

A, for Far Western analysis, recombinant PNK, Lig IIIα, Pol β, or bovine serum albumin (BSA) were run in SDS-PAGE, renatured, incubated with YB-1, and then blotted with anti-YB-1 antibody. Left panel, Coomassie staining of the corresponding proteins. B, Far Western analysis for the interaction studies between full-length or different GST-YB-1 deletion mutants (Δ6– Δ9) with Pol β (left panel) and Lig IIIα (right panel). C, Far Western analysis of the interaction between YB-1 and different C-terminal (16, 14, and 8 kDa) or N-terminal (31 and 22 kDa) deletion mutants of Pol β using anti-YB-1 antibody. Schematic representations of the Pol β domains are shown in the bottom panel.

FIGURE 5. Interaction between NEIL2 and YB-1 under oxidative stress

A, HCT116N cells were treated with UVA or glucose oxidase to induce oxidative stress for different times (as indicated in the figure) in the presence or absence of NAC, and ROS generation was evaluated by measuring the fluorescence of oxidized H₂DCFDA. Riboflavin was used as a photosensitizer. B, nuclear extracts were prepared from HCT116N cells at different time points after UVA treatment (as indicated in the left panel) or glucose oxidase (GO) treatment (right panel), immunoprecipitated with anti-FLAG antibody-conjugated beads (Sigma), and analyzed by Western blot using anti-YB-1 or anti-FLAG antibody. Lower panel, quantification of the Western blot data (YB-1 bands, lanes 1–10) after normalization of NEIL2-FLAG bands. C, HCT116N cells were treated with UVA or glucose oxidase for the indicated times and the YB-1 levels were measured and quantified (lower panel) in nuclear extract or whole cell lysate on Western blots. Lamin B and α-tubulin were used as the loading control for nuclear extract and whole cell lysate respectively.

FIGURE 6. Incision activity of NEIL2-FLAG immunocomplex after oxidative stress

Incision activity of NEIL2-FLAG immunocomplex from UVA-treated cells (lanes 3–8) on 5-OHU containing ³²P-labeled bubble substrate. NAC was used as a ROS scavenger (lanes 5, 8, and 12). Glucose oxidase (GO) was used as a positive control for ROS generation (lane 11). S, substrate; P, product.

FIGURE 7. Incision activity of NEIL2-FLAG immunocomplex after YB-1 down-regulation

A, upper panel, real time RT-PCR for optimization of YB-1 down-regulation with different concentrations of YB-1 siRNA (20–80 nm) after 24 or 48 h treatment. Lower panel, Western analysis for YB-1 expression at the indicated time points and α tubulin as a loading control. B, incision activity of NEIL2-FLAG immunocomplex from YB-1 down-regulated, UVA-treated cells (left panel). Expression of NEIL2-FLAG and YB-1 in nuclear extract and their amount in NEIL2-FLAG IP from the nuclear extract (right panel). C, effect of Pol β (left panel) and Lig IIIα (right panel) on the incision activity of NEIL2.