Study of the recognition of G-quadruplex DNA by human ORC protein

概要

Study of the recognition of G-quadruplex DNA by human ORC
protein
Afaf Sobhi Mohamed Mahmoud Eladl
DNA replication starts from multiple chromosomal loci called replication origins.
Origin recognition complex (ORC) binds to a replication origin in eukaryotic DNA and
recruit other replication factors. ORC composed of six subunits (ORC1-6) is highly
conserved in all eukaryotes and plays important role in the initiation of DNA replication. It
is well-known that the ORC of Saccharomyces cerevisiae recognizes an origin through
sequence-specifically binding to autonomously replicating sequences. However, human
ORC (hORC) binds to a replication origin without sequence specificity and how hORC
recognizes the origin remains unknown.

Previous genome-wide studies revealed that guanine (G)-rich sequences, forming Gquadruplex (G4) structure, are present in most replication origins of fly, mouse, and human
cells. So far, it was revealed that the deletion of G-rich sequences causes functional
impairment of replication origins and the insertion of G-rich sequences creates a new
replication origin. These results suggested that the G4 structure plays a critical role in the
initiation of DNA replication.
Previously, we found that the region comprising residues 413–511 of human ORC
subunit 1, hORC1413–511, is responsible for binding to G-rich DNAs, which form a G4
structure in the absence of hORC1413–511. Here, we investigated the interaction of hORC1413511

with various DNAs derived from human c-myc promoter and telomere region, having G4

structures. mtPu22 and mtPu19, were revealed to fold only into a parallel-type G4 structure
while teloDNA was reported to be the (3+1)-type G4 structure.

In this study, we showed the binding affinity to G4 relative to double-stranded DNAs
(dsDNAs) using fluorescence anisotropy (FA). Then, we demonstrated the conformational
changes of hORC1413–511 and G4-DNA in response to their interacting binding by circular
dichroism (CD) and nuclear magnetic resonance (NMR). Moreover, the binding sites for that
interaction had been identified.
In chapter 1, firstly, a general introduction about ORC protein and its structure were
given. Then, the diverse physiological and pathological roles of hORC1 were introduced.
Finally, the background and the aim of this study were described.
In chapter 2, bindings of hORC1413–511 to G-rich DNAs were characterized by
monitoring the change in the FA of fluorescein (FAM)-labeled G-rich DNAs upon titration
with hORC1413–511. Binding curves indicated that hORC1413–511 can bind to all the studied
G4-DNAs stronger than its binding to dsDNA.We further identified the necessity of the G4
structure for the binding. Data showed that G4 structure of DNA is critical for strong binding
of hORC1413–511.Then, the recorded CD spectra of G-rich DNAs showed that G- rich DNAs
retain the G4 structure even after binding with hORC1413–511 and the binding of hORC1413–
511

does not unfold the G4 structure. These results were confirmed by the retention of the G4

structure in each complex using NMR-detected hydrogen-deuterium (H/D) exchange
experiments. By the analysis of NMR chemical shift perturbation (CSP), we revealed that
the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413–
511

.
In chapter 3, first, we showed the resonance assignment and data deposition which

was indicated by backbone 1HN, 15N, 13Cα, 13Cβ and 13C’ chemical shifts of hORC1413-511.
Then, the structural characteristics of the hORC1413-511 in its free were investigated. CD and
NMR studies indicated that hORC1413-511 is intrinsically disordered containing a short helical
region in the free form.
The program TALOS+ was used to predict the secondary structure of hORC1413-511
from the assigned backbone 1HN, 15N, 13Cα, 13Cβ and 13C’ chemical shifts. The correlation
observed between the predicted secondary structure by NMR data suggests confidence in the

obtained resonance assignment. Finally, the predicted three dimensional model of hORC1
for alpha fold structure was shown.
In chapter 4, the structural characteristics of the hORC1413-511 in its complexed forms
with G4-DNAs were investigated. NMR studies indicated that hORC1413-511 retains the same
structural properties upon complex formation. The CSP analysis indicated that the basic
residues, arginine and lysine, and the polar residues, serine and threonine, are involved in the
G4-DNA binding. Interestingly, the results revealed that hORC1413-511 binds to both paralleland 3+1-type G4-DNAs using the same residues, thereby in the same manner.
In chapter 5, a general conclusion as to the research background, results outcomes,
and importance of the study was given.
In this study we demonstrated the conformational change of hORC1413–511 upon
binding G4-DNAs sequences. The identified mode through which hORC1413–511 interacts
with the guanine residues located in the external G-tetrads may rationalize the foldindependent interaction. For G4 binding, we assumed a stacking interaction between the
guanine bases and an aromatic ring of F511, which is an only aromatic residue of hORC1413–
511

and/or the guanidium groups of the arginine residues of hORC1413–511.
For hORC1413–511 binding, we revealed that both electrostatic interaction and

hydrogen bond formation are involved. Our studies suggested that hORC1 uses its
intrinsically disordered G4-binding region to play a crucial role in the recognition of the
parallel-type and 3+1-type G4 structures at the replication origins.
In conclusion, our results indicate a possible role of various G4 structures in
replication origins recognition by hORC1413–511, which is a hot topic of study due its direct
implication in the cancer diseases. ...