DNA replication process is tightly controlled in space and time. The binding of Origin Recognition Complex (ORC) to origins of replication serves as a scaffold for the assembly of a multi-protein replicative complex to initiate DNA replication in eukaryotic cells. The Orc6 protein, which is the smallest and least conserved ORC subunit, shows no structural similarity to other five subunits of ORC. Here, XU Naining, LIU Changdong and I determined the full-length structure of human Orc6 by NMR spectroscopy and demonstrated that hOrc6 binds to AT-rich DNA preferably. Moreover, two new binding sites in the middle region and C-terminal domain were identified in this study. Furthermore, XU Naining, LIU Changdong and I proved that hOrc6 mutants with defective DNA binding can abolish DNA...[ Read more ]

DNA replication process is tightly controlled in space and time. The binding of Origin Recognition Complex (ORC) to origins of replication serves as a scaffold for the assembly of a multi-protein replicative complex to initiate DNA replication in eukaryotic cells. The Orc6 protein, which is the smallest and least conserved ORC subunit, shows no structural similarity to other five subunits of ORC. Here, XU Naining, LIU Changdong and I determined the full-length structure of human Orc6 by NMR spectroscopy and demonstrated that hOrc6 binds to AT-rich DNA preferably. Moreover, two new binding sites in the middle region and C-terminal domain were identified in this study. Furthermore, XU Naining, LIU Changdong and I proved that hOrc6 mutants with defective DNA binding can abolish DNA replication in vivo. Based on these findings, a model of Orc6 binding to 10bp dsDNA is produced. These results strongly indicated that Orc6 protein is an essential part of the origin recognition complex in DNA replication.

Phase separation of protein/nucleic acid is crucial in many cellular processes in nucleus. Basically, all the proteins that can undergo phase separation contain intrinsically disordered regions involved in multivalent interactions. Many proteins in DNA replication have intrinsically disordered regions with particularly high propensity for phase separation. However, little is known about the liquid-liquid phase separation in human DNA replication machinery. Here, XU Naining, LIU Changdong and I showed that hOrc6 can form phase separation in the presence of DNA.Strikingly, the disordered C-terminal is essential for hOrc6-DNA phase separation. In addition, hOrc6 mutants with defective DNA binding was able to abolish DNA induced hOrc6 phase separation as well as DNA replication. Furthermore, hOrc6 performed puncta-like pattern in cell nucleus while the mutants cannot. Moreover, the DNA replication regulatory protein Geminin could be selectively recruited into the hOrc6-DNA droplets. These results strongly indicated that DNA replication in human requires the formation of large-scale droplets at replication origins by recruiting other pre-replication proteins to perform effective biochemical functions.