Korean J Physiol Pharmacol.  2023 Sep;27(5):427-436. 10.4196/kjpp.2023.27.5.427.

Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage

• Kim JH
• Patel R

• KMID: 2545533
• DOI: http://doi.org/10.4196/kjpp.2023.27.5.427

Abstract

Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares high sequence homology with Mad2, the mitotic checkpoint protein. Previously, we demonstrated the involvement of Mad2B in the cisplatininduced DNA damage response. In this study, we extend our findings to show that Mad2B is recruited to sites of DNA damage in human cancer cells in response to cisplatin treatment. We found that in undamaged cells, Mad2B exists in a complex with Polζ-Rev1 and the APC/C subunit Cdc27. Following cisplatin-induced DNA damage, we observed an increase in the recruitment of Mad2B and Cdc20 (the activators of the APC/C), to the complex. The involvement of Mad2B-Cdc20-APC/C during DNA damage has not been reported before and suggests that the APC/C is activated following cisplatin-induced DNA damage. Using an in vitro ubiquitination assay, our data confirmed Mad2B-dependent activation of APC/C in cisplatin-treated cells. Mad2B may act as an accelerator for APC/C activation during DNA damage response. Our data strongly suggest a role for Mad2B-APC/C-Cdc20 in the ubiquitination of proteins involved in the DNA damage response.

Keyword

Anaphase-promoting; complex-cyclosome; Cell division cycle 20; DNA damage; DNA polymerase ζ; Mad2B

Figure

• 

  Fig. 1 Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) levels increase in HeLa cell after cisplatin-induced DNA damage. Activation of the proliferating cell nuclear antigen (PCNA) and phosphorylation of H2A.X following cisplatin treatment (A, B). HeLa cells were treated with cisplatin (100 μM) for 12 h. PCNA (A) and γH2A.X (B) were stained with anti-PCNA and anti-pH2AX (S139) (red) antibodies, respectively. DNA was stained with Hoechst 33342 (blue). Cells were observed using a Nikon inverted fluorescence microscope (TE300). Approximately 400 cells were counted. White bar, control; Black bar, cisplatin. Scale bar, 10 μm. (C) Mad2B binding to chromatin was increased following cisplatin treatment. Exponentially growing HeLa cells were treated with cisplatin (50 μM) for 12 h and the cytoplasmic and chromatin-associated protein fractions were immunoblotted with antibodies against Mad2B, histone H3, and α-tubulin. (D) Cisplatin (50 μM) treatment increased the expression of Mad2B as determined via Western blotting of whole HeLa cell extracts (n = 3). Quantitation of the data shown in lower panel. Each bar represents the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 using a two-tailed t-test).

• 

  Fig. 2 Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binds to cell division cycle (Cdc)-20 following cisplatin addition. (A) HeLa cells were transfected with HA-Mad2B for 48 h. Cdc20 was immunoprecipitated from the transfected cells following cisplatin treatment (50 μM for 12 h). Immunoprecipitates were immunoblotted with antibodies against HA (middle panel), Cdc20 (upper panel), and γ-tubulin (lower panel). (B) HA-Mad2B co-localizes with Cdc20 following cisplatin addition. HeLa cells were co-transfected with Myc-Cdc20 and HA-Mad2B for 24 h and then treated with cisplatin (50 μM) for 24 h. Cells were stained for HA-Mad2B (green), DNA (blue), and Myc-Cdc20 (red). Scale bar, 10 μm. (C) Glutathione S-transferase (GST)-pull-down assay. Cell lysates were prepared from exponentially growing cells (interphase) treated with cisplatin (50 μM) for 18 h. Purified GST-Mad2B proteins (0.2–1 μM) were added to the cell lysates. Samples were incubated before the addition of glutathione-sepharose beads. Proteins bound to the beads were immunoblotted with the anti-Cdc20 antibody. As a negative control, lysis buffer was used for the pull-down assay (buffer only). As a positive control, total cell lysates (5% input) were used for identifying Cdc20 on the Western blots (WB). This result is representative of three independent experiments. IP, immunoprecipitation; HA, hemagglutinin.

• 

  Fig. 3 Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binding to cell division cycle (Cdc)-27 is increased following cisplatin-induced DNA damage. (A) HeLa cells were treated with cisplatin (50 μM) for 18 h. Cdc27 was immunoprecipitated and immunoblotted with the anti-Mad2B (upper panel) and anti-Cdc27 (low panel) antibodies. (B) Cdc27 was immunoprecipitated and immunoblotted with the anti-HA (upper panel), anti-Cdc27 (lower panel), and anti-α-tubulin (middle panel) antibodies. For control immunoprecipitation (IP), Cdc27 antibody was incubated with the lysis buffer alone. Asterisk indicates a non-specific band (IgG heavy chain). (C) HeLa cells were transfected with HA-Mad2B and treated with cisplatin (50 μM) for 18 h. Cells were stained with the anti-HA (green) and anti-Cdc27 (red) antibodies. DNA was stained with Hoechst 33342 (blue). Scale bar, 5 μm. Molecular weight markers (kD) are indicated on the left of each Western blot (WB). HA, hemagglutinin.

• 

  Fig. 4 Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) forms a complex with Rev3 and Rev1 that binds to cell division cycle (Cdc)-27 following cisplatin-induced DNA damage. HEK293 cells were co-transfected with HA-Mad2B and either FLAG-tagged hRev 1 (a.a. 907–1251) (A) or FLAG-tagged hRev 3 (a.a. 1665–2070) (B). After transfection for 24 h, the cells were treated with cisplatin (50 μM) for 18 h. Cells were then lysed and HA-Mad2B immunoprecipitated and immunoblotted with anti-HA, anti-FLAG, and anti-γ-tubulin antibodies. Control immunoprecipitates were incubated with the lysis buffer alone. (C, D) HEK293 cells were co-transfected with either HA-Mad2B and FLAG-tagged hRev1 (a.a. 907–1251) or HA-Mad2B and hRev3 (a.a. 1665–2070). Cells were then lysed and Cdc27 was immunoprecipitated from the cell lysates. Immunoprecipitates were Western blotted with anti-FLAG (upper), anti-HA (middle), and anti-Cdc27 (lower) antibodies. For control immunoprecipitates, the Cdc27 antibody was incubated with the lysis buffer alone. Molecular weight markers (kDa) are indicated on the left of each Western blot (WB). Asterisk indicates the non-specific band. IP, immunoprecipitation; HA, hemagglutinin.

• 

  Fig. 5 Activity of the anaphase-promoting complex or cyclosome (APC/C) in response to DNA damage with and without mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) via an in vitro ubiquitination assay. HeLa cells were either transfected with the control small interfering RNA (siRNA) oligonucleotide (10 nM) or Mad2B siRNA oligonucleotide (10 nM). Then, 24 h post-transfection, the cells were treated with 50 μM cisplatin for varying time periods (0–12 h). APC/C was immunopurified from the control and Mad2B-depleted cells. As a positive control, APC/C was immunopurified from mitotic HeLa cells. Immunopurified APC/C was then normalized by quantification of the protein concentration. A ubiquitination mixture containing E1, E2, ubiquitin, ATP, Myc-tagged Cyclin B N-terminal residues 1-90 (Myc-CycB N90) as a substrate, and 1.5 μM of Cdc20 protein were added. Ubiquitinated Cyclin B (CycB N90) was analyzed via Western blotting with an anti-Myc tag antibody and the quantitated data of ubiquitinated CycB is shown in the lower panel. Lane 1, APC/C without Cdc20 addition (negative control); Lanes 2, 3, and 4, transfection with the control siRNA oligonucleotide; Lanes 5, 6, and 7, transfection with Mad2B siRNA oligonucleotide; Lane 8, APC/C from Nocodazole (0.2 μg/ml for 16 h)-arrested HeLa cells.

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