RESUMO
Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.
Assuntos
DNA Helicases , Replicação do DNA , Animais , DNA/química , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Mamíferos/genéticaRESUMO
The dynamics and efficiency of photoinduced charge transport has been investigated in DNA capped hairpins possessing a stilbenedicarboxamide (Sa) hole donor and stilbenediether (Sd) hole acceptor separated by DNA G-quadruplex structures possessing 2-to-4 tetrads by means of femtosecond and nanosecond transient absorption spectroscopy with global analysis. The results for the quadruplex structures are compared with those for the corresponding duplex structures having G-C base pairs in place of the G-tetrads. Following photoinduced charge separation to form a contact radical ion pair, hole transport to form the Sa-â¢/Sd+⢠charge-separated state is slower but more efficient for the quadruplex vs duplex structures. Thus, the G-quadruplex serves as an effective conduit for positive charge rather than as a hole trap when inserted into a duplex, as previously postulated.
Assuntos
DNA/química , Quadruplex G , Éteres/química , Estrutura Molecular , Processos Fotoquímicos , Estilbenos/química , TermodinâmicaRESUMO
The electronic excited states populated upon absorption of UV photons by DNA are extensively studied in relation to the UV-induced damage to the genetic code. Here, we report a new unexpected relaxation pathway in adenine-thymine double-stranded structures (AT)n . Fluorescence measurements on (AT)n hairpins (six and ten base pairs) and duplexes (20 and 2000 base pairs) reveal the existence of an emission band peaking at approximately 320â nm and decaying on the nanosecond time scale. Time-dependent (TD)-DFT calculations, performed for two base pairs and exploring various relaxation pathways, allow the assignment of this emission band to excited states resulting from mixing between Frenkel excitons and adenine-to-thymine charge-transfer states. Emission from such high-energy long-lived mixed (HELM) states is in agreement with their fluorescence anisotropy (0.03), which is lower than that expected for π-π* states (≥0.1). An increase in the size of the system quenches π-π* fluorescence while enhancing HELM fluorescence. The latter process varies linearly with the hypochromism of the absorption spectra, both depending on the coupling between π-π* and charge-transfer states. Subsequently, we identify the common features between the HELM states of (AT)n structures with those reported previously for alternating (GC)n : high emission energy, low fluorescence anisotropy, nanosecond lifetimes, and sensitivity to conformational disorder. These features are also detected for calf thymus DNA in which HELM states could evolve toward reactive π-π* states, giving rise to delayed fluorescence.
Assuntos
Adenina/química , Citosina/química , DNA/química , Oligonucleotídeos/síntese química , Timina/química , Animais , Bovinos , DNA/metabolismo , Transferência de Energia , Oligonucleotídeos/química , Teoria Quântica , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
Molecular structures that direct charge transport in two or three dimensions possess some of the essential functionality of electrical switches and gates. We use theory, modeling, and simulation to explore the conformational dynamics of DNA three-way junctions (TWJs) that may control the flow of charge through these structures. Molecular dynamics simulations and quantum calculations indicate that DNA TWJs undergo dynamic interconversion among "well stacked" conformations on the time scale of nanoseconds, a feature that makes the junctions very different from linear DNA duplexes. The studies further indicate that this conformational gating would control charge flow through these TWJs, distinguishing them from conventional (larger size scale) gated devices. Simulations also find that structures with polyethylene glycol linking groups ("extenders") lock conformations that favor CT for 25 ns or more. The simulations explain the kinetics observed experimentally in TWJs and rationalize their transport properties compared with double-stranded DNA.
Assuntos
DNA/química , Simulação de Dinâmica Molecular/estatística & dados numéricosRESUMO
DNA-based molecular electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across DNA three-way junction (3WJ) have yet to be determined. Here, we present an investigation of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and molecular dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently populated hole carrier nucleobases into better aligned geometries on the nanosecond time scale, thus modulating the π-π electronic coupling along the base pair sequence.
Assuntos
DNA/química , Pareamento de Bases , Transporte de Elétrons , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Análise EspectralRESUMO
DNA hairpins having both a tethered anthraquinone (Aq) end-capping group and a perylenediimide (PDI) base surrogate were synthesized, wherein Aq and PDI are each separated from a G-C base pair hole trap by A-T and I-C base pairs (G = guanine, A = adenine, T= thymine, C = cytosine, I = inosine). Selective photoexcitation of PDI at 532 nm generates a singlet radical ion pair (RP), (1)(G(+â¢)-PDI(-â¢)), while selective photoexcitation of Aq at 355 nm generates the corresponding triplet RP, (3)(G(+â¢)-Aq(-â¢)). Subsequent radical pair intersystem crossing within these spin-correlated RPs leads to mixed spin states that exhibit spin-polarized, time-resolved EPR spectra in which the singlet- and triplet-initiated RPs have opposite phases. These results demonstrate that a carefully designed DNA hairpin can serve as a photodriven molecular spin switch based on wavelength-selective formation of the singlet or triplet RP without significant competition from undesired energy transfer processes.
Assuntos
DNA/química , Sequências Repetidas Invertidas/efeitos da radiação , Luz , Sequência de Bases , DNA/genética , Lasers , TemperaturaRESUMO
Double-stranded DNA conjugates with the sequence (dA)10·(dT)10 and hexaethylene glycol linkers at one end (hairpin) or both ends (dumbbell) were studied in buffer solution by deep UV femtosecond transient absorption spectroscopy. These covalently constrained duplexes have greatly enhanced thermal stability compared to A·T duplex oligonucleotides that lack linkers. The conjugates eliminate the slipped-strand and end-frayed structures that form readily in unlinked (dA)n·(dT)n sequences, allowing the excited-state dynamics of stacked A·T base pairs to be observed without interference from structures with stacking or pairing defects. Transient absorption signals show that subpicosecond internal conversion to the electronic ground state takes place in addition to the formation of long-lived excited states having lifetimes of approximately 70 ps. Watson-Crick base-pairing slows the rate of vibrational cooling compared to monomeric bases or single-stranded DNA, possibly by reducing the total number of solute-solvent hydrogen bonds. Long-lived excited states in intact A·T base pairs decay several times more quickly than long-lived excited states observed in single-stranded (dA)n sequences. These results show that base-pairing can measurably affect nonradiative decay pathways in A·T duplexes.
Assuntos
Adenina/química , DNA/química , Etilenoglicóis/química , Teoria Quântica , Timina/química , Pareamento de Bases , Conformação de Ácido NucleicoRESUMO
Guanine-cytosine hairpins, containing a hexaethylene glycol bridge, are studied by steady-state fluorescence spectroscopy and time-correlated single photon counting; their properties are compared to those of duplexes with the same sequence. It is shown that, both in hairpins and in duplexes, base pairing induces quenching of the ππ* fluorescence, the quantum yield decreasing by at least two orders of magnitude. When the size of the systems increases from two to ten base pairs, a fluorescent component decaying on the nanosecond time-scale appears at energy higher than that stemming from the bright states of non-interacting mono-nucleotides (ca. 330 nm). For ten base pairs, this new fluorescence forms a well-defined band peaking at 305 nm. Its intensity is about 20% higher for the hairpin compared to the duplex. Its position (red-shifted by 1600 cm(-1)) and width (broader by 1800 cm(-1) FWHM) differ from those observed for large duplexes containing 1000 base pairs, suggesting the involvement of electronic coupling. Fluorescence anisotropy reveals that the excited states responsible for high energy emission are not populated directly upon photon absorption but are reached during a relaxation process. They are assigned to charge transfer states. According to the emerging picture, the amplitude of conformational motions determines whether instantaneous deactivation to the ground state or emission from charge transfer states will take place, while ππ* fluorescence is associated to imperfect base-pairing.
Assuntos
Pareamento de Bases , Citosina/química , Guanina/química , Sequência de Bases , Elétrons , Etilenoglicóis/química , Teoria Quântica , Espectrometria de FluorescênciaRESUMO
We report here the effect of replacing one or both of the purine or pyrimidine blocks of a diblock stilbene donor-acceptor capped hairpin with locked nucleic acid (LNA) bases on the dynamics and efficiency of hole transport. The structures of the DNA and LNA:DNA hybrids are tentatively assigned to B- or A-type structures on the basis of their circular dichroism spectra. Replacing the bases in either the A-block or the G-block of the diblock DNA hairpin with LNA bases results in a modest decrease in the base-to-base hopping rate constant and quantum yield for charge separation. Somewhat larger decreases are observed when all of the purine or pyrimidine bases are replaced by LNA bases.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , DNA/genética , Sequências Repetidas Invertidas , Simulação de Dinâmica Molecular , Hibridização de Ácido Nucleico , Oligonucleotídeos/genéticaRESUMO
A series of DNA hairpins (AqGn) possessing a tethered anthraquinone (Aq) end-capping group were synthesized in which the distance between the Aq and a guanine-cytosine (G-C) base pair was systematically varied by changing the number (n - 1) of adenine-thymine (A-T) base pairs between them. The photophysics and photochemistry of these hairpins were investigated using nanosecond transient absorption and time-resolved electron paramagnetic resonance (TREPR) spectroscopy. Upon photoexcitation, (1*)Aq undergoes rapid intersystem crossing to yield (3*)Aq, which is capable of oxidizing purine nucleobases resulting in the formation of (3)(Aq(-â¢)Gn(+â¢)). All (3)(Aq(-â¢)Gn(+â¢)) radical ion pairs exhibit asymmetric TREPR spectra with an electron spin polarization phase pattern of absorption and enhanced emission (A/E) due to their different triplet spin sublevel populations, which are derived from the corresponding non-Boltzmann spin sublevel populations of the (3*)Aq precursor. The TREPR spectra of the (3)(Aq(-â¢)Gn(+â¢)) radical ion pairs depend strongly on their spin-spin dipolar interaction and weakly on their spin-spin exchange coupling. The anisotropy of (3)(Aq(-â¢)Gn(+â¢)) makes it possible to determine that the π systems of Aq(-â¢) and G(+â¢) within the radical ion pair are parallel to one another. Charge recombination of the long-lived (3)(Aq(-â¢)Gn(+â¢)) radical ion pair displays an unusual bimodal distance dependence that results from a change in the rate-determining step for charge recombination from radical pair intersystem crossing for n < 4 to coherent superexchange for n > 4.
Assuntos
Antraquinonas/química , Pareamento de Bases , DNA/química , Sequência de Bases , Espectroscopia de Ressonância de Spin Eletrônica , Guanina/química , Íons/química , Modelos Moleculares , FotoquímicaRESUMO
Transport of positive charge or holes in DNA occurs via a thermally activated multi-step hopping mechanism. The fastest hopping rates reported to date are those for repeating poly(purine) sequences in which hopping occurs via a random walk mechanism with rate constants of k(hop) = 4.3 × 10(9) s(-1) for poly(dG) and 1.2 × 10(9) s(-1) for poly(dA). We report here the dynamics of charge separation in DNA conjugates possessing repeating 7-deazaadenine (dzA) sequences. These data provide an estimated value of k(hop) = 4.2 × 10(10) s(-1) for poly(dzA), an order of magnitude faster than for poly(dG).
Assuntos
DNA/química , Estrutura Molecular , Purinas/químicaRESUMO
We report the measurement of distance- and temperature-dependent rate constants for charge separation in capped hairpins in which a stilbene hole acceptor and hole donor are separated by A(3)G(n) diblock polypurine sequences consisting of 3 adenines and 1-19 guanines. The longer diblock systems obey the simplest model for an unbiased random walk, providing a direct measurement of k(hop) = 4.3 × 10(9) s(-1) for a single reversible G-to-G hole hopping step, somewhat faster than the value of 1.2 × 10(9) s(-1) calculated for A-tract hole hopping. The temperature dependence for hopping in A(3)G(13) provides values of E(act) = 2.8 kcal/mol and A = 7 × 10(9) s(-1), consistent with a weakly activated, conformationally gated process.
Assuntos
DNA/química , Guanina/química , Cinética , Análise Espectral , TemperaturaRESUMO
The photophysical and photochemical behavior of a series of hairpin-forming DNA conjugates possessing a 5'-tethered pyrenecarboxamide chromophore and one or two bromouracil bases has been investigated. Quenching of the pyrene fluorescence and transient absorption spectra characteristic of the pyrene cation radical are observed only when bromouracil is located at the first or second base pair position nearest to the point of pyrene attachment. These observations are consistent with an intercalated structure for these conjugates in which pyrene is adjacent to the second base pair. Selective quenching of singlet pyrene by bromouracil but not by thymine is consistent with the free energy for charge separation estimated using Weller's equation. Low quantum yields for loss of bromide when bromouracil is not adjacent to pyrene are attributed to inefficient charge separation via either a multistep electron transport or a single-step superexchange mechanism. Quantum yields are only weakly dependent upon the distance between pyrene and bromouracil, as expected for a multistep electron transport mechanism. Loss of bromide from conjugates possessing two bromouracils occurs sequentially. For adjacent bromouracils, competitive loss of bromide from both bromouracils is observed, whereas for nonadjacent bromouracils loss of bromide from the proximal bromouracil occurs prior to any loss from the distal bromouracil, consistent with a slower rate constant for electron transport vs loss of bromide.
Assuntos
Bromouracila/química , DNA/química , Pirenos/química , Pareamento de Bases , Transporte de Elétrons , Halogenação , Processos Fotoquímicos , Análise Espectral , Especificidade por SubstratoRESUMO
The efficiency of singlet and triplet charge radical ion-pair formation and the dynamics of radical-pair charge recombination in DNA-anthraquinone conjugates have been investigated by means of femtosecond time-resolved transient absorption spectroscopy. Singlet charge separation is more efficient than intersystem crossing, resulting in inefficient formation of the long-lived triplet radical ion pair. Both singlet charge separation and charge recombination are faster when guanine rather than adenine is the neighboring purine base.
Assuntos
Antraquinonas/química , DNA/química , Estrutura Molecular , Teoria QuânticaRESUMO
The realization of highly efficient photoinduced charge separation across the pi-stacked base pairs in duplex DNA remains elusive. The low efficiencies (<5%) typically observed for charge separation over a dozen or more base pairs are a consequence of slow charge transport and rapid charge recombination. We report here a significant (5-fold or greater) enhancement in the efficiency of charge separation in diblock purine oligomers consisting of two or three adenines followed by several guanines, when compared to oligomers consisting of a single purine or alternating base sequences. This approach to wire-like behavior is attributed to both slower charge recombination and faster charge transport once the charge reaches the G-block in these diblock systems.