Binding of Human MCM-BP with MCM2-7 Proteins

MCM2-7 proteins play essential roles in DNA replication in eukaryotic cells, probably by acting as a replicative DNA helicase that unwinds DNA duplexes at replication forks (Bell & Dutta, 2002; Forsburg, 2004; Masai et al., 2010). Several lines of evidences suggest that MCM2-7 hexameric complexes assembled on the replication origins are converted to an active form with the assistance of the CDC7, CDC45 and GINS complex (Moyer et al., 2006; Gambus et al., 2006). MCM-BP, which has been identified from human cells as a protein that binds to MCM6 and MCM7 proteins, has amino acids sequences homologous to MCM2-7 (Sakwe et al. 2007). The results in this report indicate that MCM-BP replaces MCM2 in MCM2-7 complex and it binds to a replication origin in HeLa cells, suggesting that the MCM complex containing the MCM-BP may play a role in the initiation of DNA replication. It has also been indicated that downregulation of MCM-BP affects chromatin binding of MCM4. Recently it has been reported that Arabidopsis thaliana ETG1, which has been identified as an E2F target gene, is a homolog of MCM-BP (Takahashi et al., 2008). ETG1 protein is required for efficient DNA replication. Depletion of ETG1 results in inhibition of DNA replication and G2 arrest. Under these conditions, the G2 checkpoint system is induced. The report by Takahashi et al. (2010) indicates that ETG1 is involved in sister chromatid cohesion which is required for post-replicative homologous recombination repair. More recently, it has been reported that Xenopus MCM-BP regulates unloading of the MCM2-7 complex from chromatin in the late S phase by interacting with MCM7 (Nishiyama et al., 2011). These evidences suggest a possibility that MCM-BP may interact with the MCM2-7 complex at the replication forks to regulate the chromatin binding of the complex. Such interaction may be required for establishment of the cohesin complex at the forks. Here we examined biochemical properties of human MCM-BP. First, we found that human MCM-BP can bind all the human MCM2-7 proteins when the MCM-BP and one of the MCM2-7 proteins are co-expressed in insect cells. However, the interaction of MCM-BP with MCM7 was mainly detected when all the MCM2-7 and MCM-BP were co-expressed at the same time. In HeLa cells, MCM-BP was mainly recovered in a Triton-soluble fraction, suggesting that it does not stably bind to chromatin. A small portion of MCM-BP in this fraction was bound to MCM4, MCM5, MCM6 and MCM7 proteins. These results suggest that MCM-BP is not a constituent of pre-RC and it exhibits its functions by interacting with