7-Deaza-2′-deoxyadenosine and 3-deaza-2′-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3′- and 5′-junctions of d(A6)·d(T6) and B-DNA

7-Deaza-2′-deoxyadenosine (1, c⁷A_d) and 3-deaza-2′-deoxyadenosine (2, c³A_d) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthsized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5′ by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c³A_d decreased the bending more than replacement by c⁷A_d. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3′-site of an d(AAAAAA)-tract whereas replacement at the 5′-site showed no significant influence [1, 2].     

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

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 G-quadruplexes (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 fluorescence-based 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.     

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3′(2′) monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M_r of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2′,3′-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5′ purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate.     

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)₂ that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)₂ associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5′-to-5′ arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3′-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.     

Walking of antitumor bifunctional trinuclear PtII complex on double-helical DNA

The trinuclear BBR3464 ([{trans-PtCl(NH₃)₂}₂µ-(trans-Pt(NH₃)₂(H₂N(CH₂)₆NH₂)₂)]⁴⁺) belongs to the polynuclear class of platinum-based anticancer agents. DNA adducts of this complex differ significantly in structure and type from those of clinically used mononuclear platinum complexes, especially, long-range (Pt, Pt) intrastrand and interstrand cross-links are formed in both 5′–5′ and 3′–3′ orientations. We show employing short oligonucleotide duplexes containing single, site-specific cross-links of BBR3464 and gel electrophoresis that in contrast to major DNA adducts of clinically used platinum complexes, under physiological conditions the coordination bonds between platinum and N7 of G residues involved in the cross-links of BBR3464 can be cleaved. This cleavage may lead to the linkage isomerization reactions between this metallodrug and double-helical DNA. Differential scanning calorimetry of duplexes containing single, site-specific cross-links of BBR3464 reveals that one of the driving forces that leads to the lability of DNA cross-links of this metallodrug is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule.     

Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA

In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil (^BrU)-substituted sequence 5′-(G/C)[A]_{n = 1,2}^BrU^BrU-3′ and proposed that a formed uracil-5-yl radical mainly abstracts the C1′ hydrogen from the 5′-side of ^BrU^BrU under 302-nm irradiation condition. In the present work, we performed photoirradiation of ^BrU-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5′-(G/C)[A]_{n = 1,2}^BrU^BrU-3′ in 302-nm irradiation of ^BrU-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5′-^BrU^BrU[A]_{n = 1,2}(G/C)-3′, which suggests that both 5′-(G/C)[A]_{n = 1,2}^BrU^BrU-3′ and 5′-^BrU^BrU[A]_{n = 1,2}(G/C)-3′ are hot-spot sequences for the formation of the uracil-5-yl radical.     

Long-range oxidative damage in duplex DNA: the effect of bulged G in a G–C tract and tandem G/A mispairs

Two series of duplex DNA oligomers were prepared having an anthraquinone derivative (AQ) covalently linked at a 5′-terminus. Irradiation of the AQ at 350 nm leads to injection of an electron hole (radical cation) into the DNA. The radical cation migrates through the DNA causing reaction primarily at G_n sequences. In one series, GA tandem mispairs are inserted between GG steps to assess the effect of the mispair on the transport of the radical cation, reaction (damage) caused by the radical cation at the mispair, and repair of the resulting damage by formamidopyrimidine DNA glycosylase (Fpg). In the second series, a bulged guanine in a G₃C₂ sequence is interposed between the GG steps. These experiments reveal that neither G/A tandem mispairs nor bulged guanines are significant barriers to long-range charge migration in DNA. The radical cation does not cause reaction at guanines in the G/A tandem mispair. Reaction does occur at the bulged guanine, but it is repaired by Fpg.     

Adapting capillary gel electrophoresis as a sensitive,high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.     

Human telomere, oncogenic promoter and 5'-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics

Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G–G–G–G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K⁺ solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by double-chain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5′-untranslated regions (5′-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand–G-quadruplex complexes.     

d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K(+) or NH(4)(+) ions, through the 5'-5' stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.     

The Last Piece in the Vitamin B1 Biosynthesis Puzzle: STRUCTURAL AND FUNCTIONAL INSIGHT INTO YEAST 4-AMINO-5-HYDROXYMETHYL-2-METHYLPYRIMIDINE PHOSPHATE (HMP-P) SYNTHASE*

Vitamin B₁ is essential for all organisms being well recognized as a necessary cofactor for key metabolic pathways such as glycolysis, and was more recently implicated in DNA damage responses. Little is known about the enzyme responsible for the formation of the pyrimidine moiety (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase). We report a structure-function study of the HMP-P synthase from yeast, THI5p. Our crystallographic structure shows that THI5p is a mix between periplasmic binding proteins and pyridoxal 5′-phosphate-dependent enzymes. Mutational and yeast complementation studies identify the key residues for HMP-P biosynthesis as well as the use of pyridoxal 5′-phosphate as a substrate rather than as a cofactor. Furthermore, we could show that iron binding to HMP-P synthase is essential for the reaction.     

New chemically reactive dsDNAs containing single internucleotide monophosphoryldithio links: reactivity of 5'-mercapto-oligodeoxyribonucleotides

Novel modified DNA duplexes with single bridging 5′-SS-monophosphoryldithio links [-OP(=O)-O^–-SS-CH₂-] were synthesized by autoligation of an oligonucleotide 3′-phosphorothioate and a 5′-mercapto-oligonucleotide previously converted to a 2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent template-dependent process under the conditions used and does not require strong binding of the reactive oligomers to the complementary strand. The modified internucleotide linkage, resembling the natural phosphodiester bond in size and charge density, is stable in water, easily undergoes thiol–disulfide exchange and can be specifically cleaved by the action of reducing reagents. DNA molecules containing an internal -OP(=O)-O^–-SS-CH₂- bridge are stable to spontaneous exchange of disulfide-linked fragments (recombination) even in the single-stranded state and are promising reagents for autocrosslinking with cysteine-containing proteins. The chemical and supramolecular properties of oligonucleotides with 5′-sulfhydryl groups were further characterized. We have shown that under the conditions of chemical ligation the 5′-SH group of the oligonucleotide has a higher reactivity towards N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than does the OH group. This autoligation, unlike disulfide bond formation, proceeds only in the presence of template oligonucleotide, necessary to provide the activated phosphate in close proximity to the SH-, OH- or phosphate function.     

Dinucleotide Sequences at the 5' Ends of Vaccinia Virus mRNA's Synthesized In Vitro

The diversity of dinucleotide sequences at the 5′ ends of vaccinia virus mRNA's was determined by a two-dimensional electrophoresis procedure. RNA labeled with S-adenosyl[methyl-³H]methionine was synthesized in vitro by enzymes present in vaccinia virus cores. The RNA, ending in m⁷G(5′)pppN^mpN−, was β-eliminated and treated with alkaline phosphatase. After digestion with RNases T₂, T₁, and A, all eight possible dinucleotides containing G^m and A^m were identified. They are, in decreasing order of abundance: G^mpUp (22%), A^mpCp (18%), G^mpAp (16%), G^mpCp (15%), A^mpAp (11%), A^mpUp (10%), A^mpGp (7%), and G^mpGp (2%).     

Unique actinomycin D binding to self-complementary d(CXYGGCCY'X'G) sequences: duplex disruption and binding to a nominally base-paired hairpin

Actinomycin D (ACTD) has been shown to bind weakly to the sequence -GGCC-, despite the presence of a GpC site. It was subsequently found, however, that d(CATGGCCATG) binds relatively well to ACTD but exhibits unusually slow association kinetics, contrary to the strong-binding -XGCY- sites. In an effort to elucidate the nature of such binding and to delineate the origin of its interesting kinetic behavior, studies have now been extended to include oligomers with the general sequence motifs of d(CXYGGCCY′X′G)₂. It was found that analogous binding characteristics are observed for these self-duplex decamers and comparative studies with progressively base-truncated oligomers from the 5′-end led to the finding that d(GGCCY′X′G) oligomers bind ACTD considerably stronger than their parent decamers and exhibit 1:1 drug/strand binding stoichiometry. Melting profiles monitored at the drug spectral region indicated additional drug binding prior to the onset of eventual complex disruptions with near identical melting temperatures for all the oligomers studied. These results are consistent with the notion that the related oligomers share a common strong binding mode of a hairpin-type, with the 3′-terminus G folding back to base-pair with the C base of GGC. A binding scheme is proposed in which the oligomers d(CXYGGCCY′X′G) exist predominantly in the duplex form and bind ACTD initially at the central GGCC weak site but subsequently disrupt to accommodate the stronger hairpin binding and thus the slow association kinetics. Such a mechanism is supported by the observation of distinct biphasic fluorescence kinetic traces in the binding of 7-amino-ACTD to these duplexes.     

The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA

The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg²⁺, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.     

Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing

The prototypical 5′-nuclease, flap endonuclease-1 (FEN1), catalyzes the essential removal of single-stranded flaps during DNA replication and repair. FEN1 hydrolyzes a specific phosphodiester bond one nucleotide into double-stranded DNA. This specificity arises from double nucleotide unpairing that places the scissile phosphate diester on active site divalent metal ions. Also related to FEN1 specificity is the helical arch, through which 5′-flaps, but not continuous DNAs, can thread. The arch contains basic residues (Lys-93 and Arg-100 in human FEN1 (hFEN1)) that are conserved by all 5′-nucleases and a cap region only present in enzymes that process DNAs with 5′ termini. Proline mutations (L97P, L111P, L130P) were introduced into the hFEN1 helical arch. Each mutation was severely detrimental to reaction. However, all proteins were at least as stable as wild-type (WT) hFEN1 and bound substrate with comparable affinity. Moreover, all mutants produced complexes with 5′-biotinylated substrate that, when captured with streptavidin, were resistant to challenge with competitor DNA. Removal of both conserved basic residues (K93A/R100A) was no more detrimental to reaction than the single mutation R100A, but much less severe than L97P. The ability of protein-Ca²⁺ to rearrange 2-aminopurine-containing substrates was monitored by low energy CD. Although L97P and K93A/R100A retained the ability to unpair substrates, the cap mutants L111P and L130P did not. Taken together, these data challenge current assumptions related to 5′-nuclease family mechanism. Conserved basic amino acids are not required for double nucleotide unpairing and appear to act cooperatively, whereas the helical cap plays an unexpected role in hFEN1-substrate rearrangement.     

Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K⁺ and in Li⁺, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.     

Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix

Very-short-patch repair (Vsr) enzymes occur in a variety of bacteria, where they initiate nucleotide excision repair of G:T mismatches arising by deamination of 5-methyl-cytosines in specific regulatory sequences. We have now determined the structure of the archetypal dcm-Vsr endonuclease from Escherichia coli bound to the cleaved authentic hemi-deaminated/hemi-methylated dcm sequence 5′-C-OH-3′ 5′-p-T-p-A-p-G-p-G-3′/3′-G-p-G-p-T-p^Me5C-p-C formed by self-assembly of a 12mer oligonucleotide into a continuous nicked DNA superhelix. The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites.     

Emulsified BMVC derivative induced filtration for G-quadruplex DNA structural separation

A novel method based on emulsion/filtration is introduced for G-quadruplex DNA structural separation. We first synthesized a lipophilic analogue of BMVC, 3,6-Bis(1-methyl-4-vinylpyridinium)-9-(12′-bromododecyl) carbazole diiodide (BMVC-12C-Br), which can form an oil-in-water (o/w) phase emulsion. Due to the binding preferences of BMVC-12C-Br emulsion to some specific DNA structures, the large emulsion (∼2 µm) bound DNA was separated from the small free DNA in the filtrate by a 0.22 µm pore size MCE membrane. This method is able to isolate the non-parallel G-quadruplexes from the parallel G-quadruplexes and the linear duplexes from both G-quadruplexes. In addition, this method allows us not only to determine the absence of the parallel G-quadruplexes of d(T₂AG₃)₄ and the presence of the parallel G-quadruplexes of d(T₂AG₃)₂ in K⁺ solution, but also to verify structural conversion from antiparallel to parallel G-quadruplexes of d[AG₃(T₂AG₃)₃] in K⁺ solution under molecular PEG condition. Moreover, this emulsion can separate the non-parallel G-quadruplexes of d(G₃CGCG₃AGGAAG₅CG₃) monomer from the parallel G-quadruplexes of its dimer in K⁺ solution. Together with NMR spectra, one can simplify the spectra for both the free DNA and the bound DNA to establish a spectrum-structure correlation for further structural analysis.