A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands
Nucleic Acids Research
A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands
Oscar Mendoza 1 2
Nassima Meriem Gueddouda 1 2
Jean-Baptiste Boul e´ 0
Anne Bourdoncle 1 3
Jean-Louis Mergny 1 2
0 CNRS, UMR7196, Mus e ́um National d'Histoire Naturelle , 75005 Paris , France
1 INSERM, U869, IECB , Pessac , France
2 University of Bordeaux, ARNA laboratory , Bordeaux , France
3 Univ. Poitiers , 40 avenue du recteur Pineau, 86000 Poitiers , France
Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as Gquadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescencebased technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5 to 3 DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.
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Helicases are a central class of enzymes found in all known
organisms. By a combination of biochemical and genetic
approaches, their activity has been shown to affect most
metabolic processes that rely on nucleic acid unwinding in
the cell, including DNA replication, transcription,
translation, DNA repair or recombination. At the molecular level,
helicases are motor proteins that move directionally along a
nucleic acid phosphodiester backbone and are able to
separate annealed nucleic acid strands using energy derived from
adenosine triphosphate (ATP) hydrolysis.
Although the majority of the known helicase enzymes
are involved in the process of unwinding duplex DNA or
RNA, some of these proteins have been known to act on less
canonical substrates like protein-nucleic acids complexes or
secondary DNA structures, in particular G-quadruplexes
(G4) (1–4). In the G4 field, helicase studies have been
at the forefront for demonstrating the formation of these
structures in vivo, through various biochemical and genetic
approaches (5–10). Some of the outstanding questions in
the field are now how specific helicases are to various
Gquadruplex structures, or what is the degree of redundancy
between different helicases in their capacity to process these
structures.
It is well documented that G4 can adopt multiple
structures and strand arrangements, which differ
thermodynamically (11). How these affect the helicase activity is currently
poorly assessed. Owing to technical limitations and to the
relative youth of this field, G-quadruplex unwinding has
been monitored for a limited number of helicases,
including members of the RecQ family (12,13), the XPD family
(FANCJ) (3) and the Pif1 family (2,14,15) and for a limited
number of substrates.
The most common assay for measuring helicase
activity in vitro employs gel electrophoresis (16).
Electrophoresis has the obvious advantage to allow the determination
of the diversity and relative abundance of the molecular
species present in the reaction. This is however a relatively
cumbersome and low-throughput technique (17,18).
Several additional methods based on fluorescence assays have
been developed to overcome some of the limitations of
electrophoresis, and to tackle real-time kinetics aspects of
helicase action (3,19). To improve tools aiming at
characterizing the activity of DNA helicases towards G4 structures,
we developed a fluorescence-based helicase assay that
allows testing the unwinding of different G4 structures by a
helicase in high throughput and in real time. In this work,
we demonstrate its application to study the activity of the
DNA helicase Pif1p, a prototypal member of the PIF1
family of DNA helicase, and for which substantial evidences
have been accumulated suggesting its activity on G4 in vitro
and in vivo (15,19,20). This assay is also used to assess the
inhibitory effect of pharmacological G4 binders on the
activity of Pif1p. To our knowledge this is the first real-time
helicase assay developed that allows, in a single experiment,
the screening of series G4 sequences under several
conditions and in the presence of G4 ligands. Moreover, this
assay should be adaptable to the specific requirements of other
helicase enzymes.
MATERIALS AND METHODS
Oligonucleotides and compounds
All oligonucleotides used in this research were purchased
from Eurogentec and stored at −20◦C as 100–200 M stock
solutions. Oligonucleotide strand concentrations were
determined by absorbance at 260 nm using the extinction
coefficients provided by the manufacturer. Sequences are
provided in Table 1.
DNA sy (...truncated)