THESIS
2020
1 online resource (xviii, 123 pages) : illustrations (some color)
Abstract
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...[
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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 Ba
2+ 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.
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