G-quadruplexes are secondary structures of nucleic acid, believed to play important roles in several essential biological events including telomere regulation, initiation of DNA replication and control of gene expression. The functions of G-quadruplex could be derived from its non-canonical structure that enable specific interaction with other biomolecules. DNA replication needs to be precisely regulated, a process heavily relying on exquisite cooperation of multiple proteins, including Cdc6, in a sequential manner on the replication origin. In human genome, G-quadruplex motifs have been frequently found near origin of DNA replication. We found that the N-terminal of Cdc6 could bind to G-quadruplex DNA, and solved the complex structure by nuclear magnetic resonance (NMR) spectroscopy. B...[ Read more ]

G-quadruplexes are secondary structures of nucleic acid, believed to play important roles in several essential biological events including telomere regulation, initiation of DNA replication and control of gene expression. The functions of G-quadruplex could be derived from its non-canonical structure that enable specific interaction with other biomolecules. DNA replication needs to be precisely regulated, a process heavily relying on exquisite cooperation of multiple proteins, including Cdc6, in a sequential manner on the replication origin. In human genome, G-quadruplex motifs have been frequently found near origin of DNA replication. We found that the N-terminal of Cdc6 could bind to G-quadruplex DNA, and solved the complex structure by nuclear magnetic resonance (NMR) spectroscopy. By biochemical and structural approaches, we characterized the interaction between the Cdc6 N-terminal and G-quadruplex DNA and analysed the individual role of the involving residues. We found the N-terminal peptide prefers binding to G-quadruplex over single strand and double strand DNA, which may provide molecular basis for recognition and loading of Cdc6 onto the replication origins.

Epstein-Barr nuclear antigen 1 (EBNA1) governs the replication and partitioning of viral genomic DNA during Epstein-Barr virus (EBV) infection. Previous studies have found that EBNA1 preferentially binds to G-quadruplex forming RNA. By NMR titration, we characterized the interaction between G-quadruplex RNA and EBNA1’s RGG motif comprising repeats of GRGRGGSG sequence. We found that two repeats are sufficient for RNA binding, and the binding motif displays preference for G-quadruplex RNA over DNA. Considering this motif itself is encoded by G-rich mRNAs, our result could provide molecular mechanism for the EBNA1’s ability to regulate its own translation. As G-quadruplex RNA is regarded as mediator for recruitment of ORC by EBNA1, the study of EBNA1 and G-quadruplex RNA might also provide structural basis for assembly of pre replicative complex (pre-RC) in EBV.

Moreover, we solved the crystal structure of G-quadruplex formed by d(G4C2)₂, a sequence present in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), annealed in Ba²⁺ solution, which displays an eight-layer tetrameric parallel conformation with two dimeric blocks stack in a 5'-to-5' manner. This de novo structure would add to the repertoire of G-quadruplex structures, especially with the core sequence of G4C2 and could provide model for structure-based virtual screening and other drug development strategies of ALS and FTD.