DNA replication is required for the proliferation of all life forms. In all eukaryotic cells, initiation of DNA replication is tightly controlled to once per cell cycle through a licensing mechanism before entering the S phase. The understanding of how DNA molecules are duplicated during each cell division cycle has been a major scientific endeavor in life sciences. Previous studies suggest that DNA replication is regulated by special DNA secondary structures known as G-quadruplex (G4). And especially, it is known that the replication origins of metazoans contain special G-rich motifs termed as origin G-rich repeated elements (OGRE) which have the potential to form G4. Recent studies also indicate the role of G4-binding protein, Telomere Repeat Binding factor 2 (TRF2), in the recruitmen...[ Read more ]

DNA replication is required for the proliferation of all life forms. In all eukaryotic cells, initiation of DNA replication is tightly controlled to once per cell cycle through a licensing mechanism before entering the S phase. The understanding of how DNA molecules are duplicated during each cell division cycle has been a major scientific endeavor in life sciences. Previous studies suggest that DNA replication is regulated by special DNA secondary structures known as G-quadruplex (G4). And especially, it is known that the replication origins of metazoans contain special G-rich motifs termed as origin G-rich repeated elements (OGRE) which have the potential to form G4. Recent studies also indicate the role of G4-binding protein, Telomere Repeat Binding factor 2 (TRF2), in the recruitment of ORC1, a subunit of origin recognition complex, to the replication origins in human telomere replication. Interestingly, the cooperative role of Epstein Barr Nuclear Antigen 1 (EBNA1) and TRF2 proteins has been demonstrated in Epstein-Barr virus (EBV) origin of plasmid replication (OriP). However, the molecular mechanisms of the composition, recognition and regulation of metazoan replication origins and the recruitment of replication machinery to these origins remain poorly understood.

In this study, we report the first NMR hybrid (3+1) G-quadruplex structure, with unique structural feature, formed by G. gallus replication origins. We found that the central region of hORC1 (413-511) can bind to G4, which may serve as a docking site for origin recognition.

We also identified the direct interactions between central region of hORC1 (413-511) and dimerization domain of TRF2 (84-287). Our in vivo study also shows the colocalization of TRF2 with ORC1 in HEK293T cells supporting the direct interaction between ORC1 and TRF2. In addition, we also identified the interaction between N-terminus of TRF2 and DNA binding and dimerization domain, DBD, of EBNA1 using GST pulldown assay and NMR titration approach. In conclusion, our findings shed light on the importance of G4 in defining DNA replication origins and DNA replication initiation. We also propose that GC-rich replication origins are recognized by ORC1/TRF2 complex. Furthermore, our results explained the mechanism of EBV (OriP) replication and provide potential targets for anticancer drug screening and design.