MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3

Abstract

The MCM proteins are essential for the initiation of DNA replication. We have isolated an MCM3-associated protein (MCM3AP) in a two-hybrid screen using MCM3. Here we demonstrate that MCM3AP is an acetyltransferase which acetylates MCM3 and that chromatin-bound MCM3 is acetylated in vivo. The MCM3 acetylase, MCM3AP, is also chromatin-bound. This study also indicates that MCM3AP contains putative acetyl CoA binding motifs conserved within the GCN5-related N-acetyltransferase superfamily. Mutation of those motifs significantly inhibits the MCM3 acetylase activity. Over-expression of MCM3AP inhibits DNA replication, whereas mutation of the acetylase motifs abolishes this effect, suggesting that acetylation plays a role in DNA replication. Taken together, we suggest that MCM3 acetylation is a novel pathway which might regulate DNA replication.

Fig. 1. In vivo acetylation of MCM3. (A) The soluble protein fraction and the structure-bound protein fraction prepared from 5 × 10⁵ asynchronous cells were analyzed by 7.5% SDS–PAGE. The proteins were blotted to PVDF membrane. The membranes were incubated with indicated antibody, respectively. The bound antibody was detected by horseradish peroxidase-labeled secondary antibody and visualized by ECL PLUS kit. (B) HeLa cell extracts prepared from 1 × 10⁷ cells were incubated with protein A–Sepharose and with anti-MCM3 antibody or normal rabbit IgG (mock). After 1 h incubation, column-bound proteins were analyzed by SDS–PAGE (7.5%) and blotted to PVDF membrane. (C) MCM3/pcDNA3.1 plasmid or mock vector was transfected to 293T cells, respectively. After culture for 20 h the cells were harvested and resuspended in hypotonic buffer containing 1% NP40, 0.5 M NaCl, and 7 mM Na butyrate. Cell extracts were incubated with a TALON column and the column was washed extensively with same buffer. Bound proteins were extracted from the column with 100 mM imidazole and 7 mM Na butyrate in hypotonic buffer and analyzed by SDS–PAGE. The analyzed proteins were blotted to PVDF membrane.

Fig. 2. Effects of cell cycle stage on MCM3 acetylation. The soluble protein fraction and the structure-bound protein fraction prepared from 5 × 10⁵ cells (for anti-acetyl Lys antibody) or from 1 × 10⁵ cells (for other antibodies) treated with nocodazole or mimosine were analyzed by 7.5% SDS–PAGE. The proteins were blotted to PVDF membrane. The membranes were incubated with the indicated antibody, respectively.

Fig. 3. MCM3AP-mediated acetylation of MCM3. (A) The HAT domain of yeast HAT1 (HAT1), human putative tumor suppressor (FUS2), human Tat interactive protein (60 kDa) (TIP60), and human P/CAF (P/CAF) were compared with MCM3AP. Conserved amino acids between HAT and MCM3AP were indicated by inverted characters. The alignment of MCM3AP was adjusted manually on the basis of classification of amino acid residues: aromatic residues (F, W and Y); negative charged residues (D, E); positive charged residues (H, K, R); aliphatic residues (I, L, and V). (B) MCM3 was incubated with MCM3AP and acetyl CoA or only one of them at 30°C. After incubation the samples were analyzed by SDS–PAGE and blotted to PVDF membrane. (C) After incubation with MCM3AP in the presence or absence of acetyl CoA, the indicated amount of MCM3 was spotted onto nitrocellulose membrane. Acetylated histone H4 peptide was spotted as a standard to evaluate the amount of acetyl moiety.

Fig. 4. Effects of mutation at amino acids included in putative acetyl CoA binding site. (A) His₆-tagged wild-type (wt) or mutant (mut, ⁴⁷¹HGAG to AAAA) MCM3AP was incubated with or without MCM3 in the presence of ¹⁴C-labeled acetyl CoA. The same amount of wt and mutant MCM3AP were examined for acetyltransferase activity under the same conditions. (B) Y190 yeast strains transfected with the indicated plasmids were cultured for 4 days at 30°C on -LWH SD plates containing 25 mM 3-AT. BD; Gal4 binding domain fusion, AD; Gal4 activation domain fusion. (C) GFP-tagged wt or mutant MCM3AP were expressed in 293T cells. The proportion of GFP positive cells that were also BrdU positive is indicated. TOTO3 positive images were used to identify non transfected cells. The extent of BrdU positive cells in TOTO3 positive but GFP negative images is also indicated as ‘not transfected’.