Four-stranded Helical Structures Research Articles

G-Quadruplex Structures Colocalize with Transcription Factories and Nuclear Speckles Surrounded by Acetylated and Dimethylated Histones H3.

G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.

Modelling the DNA topology: the effect of the loop bending on G-quadruplex stability

The guanine quadruplexes are DNA/RNA guanine-rich sequences assembled into four-stranded helical structures having important roles in gene regulation and chromosome stability. Their mechanical unfolding has been generally studied with all-atom simulations, which cannot dissect the specific interactions responsible for their cohesion. Recently, we introduced a mesoscopic model able to describe the main thermal and mechanical unfolding features of the G-quadruplex, with no distinction between the different geometrical conformations forming the structure. Under the same model we study here the contribution of the bending potential in the extra-structure bonding (loops). We demonstrate with numerical simulations that the rigidity of the loops can account for the different stabilities of different topologies, specifically the parallel and the antiparallel. In the presence of the bending, the model indicates the antiparallel configuration as the most stable G4 topology, as suggested by the experiments.

Mesoscopic model for DNA G-quadruplex unfolding

Genomes contain rare guanine-rich sequences capable of assembling into four-stranded helical structures, termed G-quadruplexes, with potential roles in gene regulation and chromosome stability. Their mechanical unfolding has only been reported to date by all-atom simulations, which cannot dissect the major physical interactions responsible for their cohesion. Here, we propose a mesoscopic model to describe both the mechanical and thermal stability of DNA G-quadruplexes, where each nucleotide of the structure, as well as each central cation located at the inner channel, is mapped onto a single bead. In this framework we are able to simulate loading rates similar to the experimental ones, which are not reachable in simulations with atomistic resolution. In this regard, we present single-molecule force-induced unfolding experiments by a high-resolution optical tweezers on a DNA telomeric sequence capable of adopting a G-quadruplex conformation. Fitting the parameters of the model to the experiments we find a correct prediction of the rupture-force kinetics and a good agreement with previous near equilibrium measurements. Since G-quadruplex unfolding dynamics is halfway in complexity between secondary nucleic acids and tertiary protein structures, our model entails a nanoscale paradigm for non-equilibrium processes in the cell.

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

A parallel stranded G-quadruplex composed of threose nucleic acid (TNA).

G-rich sequences can adopt four-stranded helical structures, called G-quadruplexes, that self-assemble around monovalent cations like sodium (Na+ ) and potassium (K+ ). Whether similar structures can be formed from xeno-nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α-l-threofuranosyl nucleic acid) to adopt a four-stranded helical structure based on a planar G-quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution-state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self-assemble into stable G-quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na+ and K+ ions, as G-quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self-assembling into stable G-quadruplex structures.

G-quadruplexes and their regulatory roles in biology.

‘If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo’ (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases.

Crystal Structure of an RNA Quadruplex Containing Inosine Tetrad: Implications for the Roles of NH 2 Group in Purine Tetrads

Polyinosinic acid has been known to adopt the four-stranded helical structure but its basic unit, inosine tetrad (I tetrad), has not been determined at the atomic level. Here we report the crystal structure of an RNA quadruplex containing an I tetrad at 1.4 Å resolution. The I tetrad has one cyclic hydrogen bond N1…O6 with the bond length of 2.7 Å. A water bridge is observed in the minor groove side of the base tetrad. Even though it is sandwiched by guanine tetrads (G tetrads), the I tetrad is buckled towards the 3′ side of the tetrad plane, which results from the different interaction strength with K ions on two sides of the tetrad plane. Comparison with both G tetrad and adenine tetrad indicates that lack of NH 2 in the C2 position makes the I tetrad prone to buckle for interactions with ligands. Two U*(G-G-G-G) base pentads are observed at the junction of the 5′ termini of two quadruplexes. The uridine residue in the base pentad is engaged in two hydrogen bonding interactions (N2(G)-H…O2(U) and O2′(G)-H…O4(U)) and a water-mediated interaction (N3(G) and N3(U)) with the G tetrad. We also discuss the roles of amino group in purine tetrads and the inter-quadruplex interactions in RNA molecules. These quadruplexes may interact with each other by stacking, groove binding and intercalation.

A Novel Yeast Gene Product, G4p1, with a Specific Affinity for Quadruplex Nucleic Acids

G4 nucleic acids are four-stranded helical structures that are formed in vitro by nucleic acids that contain guanine tracts. These structures anneal readily under physiological conditions and are unusually stable once formed. G4 nucleic acids are thought to participate in telomere function, retroviral genome dimerization, chromosome alignment during homologue pairing, and mitotic recombination, although the in vivo demonstration of these structures in any of these situations has not yet been achieved. Here we purify and characterize an activity from yeast, G4p1, which has a high and specific affinity for G4 nucleic acids. G4p1 prefers substrates containing multiple G4 domains, has an equal affinity for parallel and antiparallel G4 structures, and binds equivalently to RNA and DNA in G4 form. The Keq for G4p1 binding to a G4 DNA oligomer is 5.0 x 10(8) M-1, under near physiological conditions. G4p1 was purified and shown to derive from a 42-kDa protein (p42). We have cloned and sequenced the gene encoding p42 and show it to encode a novel protein with a region significantly homologous to bacterial methionyl-tRNA synthetase dimerization domains. We have reconstituted the G4p1 binding activity with recombinant p42 and present evidence that G4p1 is a homodimer of p42.

Crystal structure of four-stranded Oxytricha telomeric DNA.

The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

Structure of Limulus telson muscle thick filaments

Computer analysis of electron micrographs of negatively stained thick filaments isolated from the telson levator muscle of the horseshoe crab ( Limulus polyphemus) has shown that they have a four-stranded helical structure. The repeating units along each helix have a bent extended shape (measuring approximately 20 nm × 8 nm × 8 nm) and are inclined at an angle of about 30 ° to the helical path. At the resolution of this study, it was difficult to establish the exact size of the surface subunits, but our results are probably more consistent with each unit representing the two heads of a single myosin molecule rather than larger aggregates.

Tertiary structure for palindromic regions of DNA

Studies of the primary structure of chromosomal DNA have revealed an interesting structural feature. Palindromic regions (sections of double-helical DNA with an axis of 2-fold rotational symmetry) were found in DNA from eukaryotes [l-6] , prokaryotes and viruses [7-131. The length of palindromic regions varies from a few to several thousand base pairs. In the present paper the three-dimensional structure (four-stranded helix) for palindromic regions is proposed. This structure differs from the cruciform structure suggested [ 141. The possible role of the four-stranded helical structure in the formation of the tertiary structure and functioning of the DNA molecule is discussed. For a preliminary description of our structure see [ 1.51 .

Secondary structures of DNA.

The X-ray diffraction patterns of DNA do not support a four-stranded helical structure.