The assembly of pre-replicative complexes (pre-RCs) at replication origins is prerequisite for eukaryotic DNA replication. Cdt1 physically interacts with and promotes the chromatin loading of the MCM complex during pre-RC assembly. The C-terminal region of hMcm6 (a.a. 708-821) has been identified to interact with a short C-terminal region of hCdt1 (a.a. 392-471) by yeast two-hybrid analysis and co-immunoprecipitation assays. We further confirmed the interaction between the interacting domains of hCdt1 and hMcm6 by GST pull-down assay. Moreover, our functional studies show that ectopic expression of either of the interacting domains in HeLa cells resulted in defective chromatin association of the MCM complex, inhibited DNA replication and cell proliferation and led to apoptosis. These do...[ Read more ]

The assembly of pre-replicative complexes (pre-RCs) at replication origins is prerequisite for eukaryotic DNA replication. Cdt1 physically interacts with and promotes the chromatin loading of the MCM complex during pre-RC assembly. The C-terminal region of hMcm6 (a.a. 708-821) has been identified to interact with a short C-terminal region of hCdt1 (a.a. 392-471) by yeast two-hybrid analysis and co-immunoprecipitation assays. We further confirmed the interaction between the interacting domains of hCdt1 and hMcm6 by GST pull-down assay. Moreover, our functional studies show that ectopic expression of either of the interacting domains in HeLa cells resulted in defective chromatin association of the MCM complex, inhibited DNA replication and cell proliferation and led to apoptosis. These dominant negative effects indicate that the interaction between hCdt1 and hMcm6 through their interacting domains is the key for hCdt1 in facilitating the MCM hexamer to load onto chromatin for replication licensing.

Ipi3, which is required for ribosome biogenesis, was identified as a novel replication-initiation protein in budding yeast. To reveal the function of hIpi3 in DNA replication, siRNAs were used to knockdown hIpi3 in HeLa cells. We found that knockdown of hIpi3 resulted in defects in chromatin association of the MCM complex and cell cycle progression, inhibited DNA replication and cell proliferation, and induced apoptosis. Our study indicates that hIpi3 functions in DNA replication, suggesting the existence of a mechanism that coordinates DNA replication with ribosome biogenesis.

In budding yeast, histone H3 with lysine 4 trimethylation (H3K4me3) physically interacts with ORC. By examining the chromosome-wide methylation states of H3K4 by ChIP assay, we found that low levels of H3K4me3 are associated with replication origins throughout the cell cycle. Furthermore, MCM occupancy at G1 phase, measured by relative ChIP enrichment, correlates with origin efficiency, and depletion of H3K4me3 alters MCM occupancy. Our study provides a linkage between H3K4me3 and replication origins, shedding new light on the epigenetic regulation of DNA replication.