DNA replication licensing is tightly controlled to ensure that genomic DNA replication happens only once in eukaryotic cells. The six-subunit origin recognition complex (ORC) is a DNA replication initiator which localized at the replication origin for the assembly of the pre-replication complex which is important for the initiation of DNA replication. The Orc6 protein is the least conversed and the smallest subunit in ORC and has a different structure to the Orc1-5 subunits, however, it is an essential element in ORC for performing the replicative function of the ORC and also involved in other cell cycle regulation. The interaction between human Orc6 (hOrc6) and DNA was studied with NMR spectroscopy which we found the middle region and a IDRs in C-terminal, residues 95-207, play an impo...[ Read more ]

DNA replication licensing is tightly controlled to ensure that genomic DNA replication happens only once in eukaryotic cells. The six-subunit origin recognition complex (ORC) is a DNA replication initiator which localized at the replication origin for the assembly of the pre-replication complex which is important for the initiation of DNA replication. The Orc6 protein is the least conversed and the smallest subunit in ORC and has a different structure to the Orc1-5 subunits, however, it is an essential element in ORC for performing the replicative function of the ORC and also involved in other cell cycle regulation. The interaction between human Orc6 (hOrc6) and DNA was studied with NMR spectroscopy which we found the middle region and a IDRs in C-terminal, residues 95-207, play an important role for DNA binding, which this region was named as hOrc6 DNA binding domain (hOrc6-DBD). Furthermore, we found the hOrc6 protein undergo liquid-liquid phase separation when induced by double-stranded DNA (dsDNA) but not G-quadruplex DNA. The binding of dsDNA is essential for the phase separation of hOrc6 and we indicated the hOrc6-DBD and the IDRs adjacent to DBD, residues 208-221, are both important for forming phase-separated droplet. We also found the Meier- Gorlin Syndrome (MGS) related hOrc6 mutation would abolish the dsDNA binding and affect the phase formation efficiency. The ability of selective recruitment of DNA replication regulation factor, Geminin, and initiation factor, MCM2-7, by the hOrc6 phase-separated droplet was showed in this study. These results strongly imply liquid-liquid phase separation of Orc6 at the origin play a critical role on DNA replication for recruitment of protein involve in re-replication complex formation to perform effective biochemical functions.