Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy

[EN] 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 pi pi(star) 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 pi pi(star) fluorescence is associated to imperfect base-pairing.The French Agency for Research (ANR-10-BLAN-0809-01), the Conselleria de Educacion-Generalitat Valenciana (VALi+D program to I.V., N° 2010033) and the US Department of Energy, Office of Basic Energy Sciences (grant no. DE-FG02-96ER14604 to F.D.L.) are acknowledged for financial support.Brazard, J.; Thazhathveetil, A.; Vayá Pérez, I.; Lewis, F.; Gustavsson, T.; Markovitsi, D. (2013). Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy. Photochemical & Photobiological Sciences Photochemical and Photobiological Sciences. 12(8):1453-1459. https://doi.org/10.1039/c3pp50088hS14531459128Bouvier, B., Dognon, J.-P., Lavery, R., Markovitsi, D., Millié, P., Onidas, D., & Zakrzewska, K. (2003). Influence of Conformational Dynamics on the Exciton States of DNA Oligomers. The Journal of Physical Chemistry B, 107(48), 13512-13522. doi:10.1021/jp036164uMezzina, E., Mariani, P., Itri, R., Masiero, S., Pieraccini, S., Spada, G. P., … Gottarelli, G. (2001). The Self-Assembly of a Lipophilic Guanosine Nucleoside into Polymeric Columnar Aggregates: The Nucleoside Structure Contains Sufficient Information To Drive the Process towards a Strikingly Regular Polymer. 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DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Eukaryotic replisomes are multiprotein complexes. Here the authors reveal two distinct stressed replisomes, associated with DONSON and FANCM, displaying a bias in replication timing and chromatin domain

Photoselective DNA Hairpin Spin Switches

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^+•-PDI^–•), while selective photoexcitation of Aq at 355 nm generates the corresponding triplet RP, ³(G^+•-Aq^–•). 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

Ultrafast Excited-State Dynamics in Hexaethyleneglycol-Linked DNA Homoduplexes Made of A·T Base Pairs

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)_<i>n</i>·(dT)_<i>n</i> 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)_<i>n</i> sequences. These results show that base-pairing can measurably affect nonradiative decay pathways in A·T duplexes

The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks

The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single molecule technique for visualizing encounters of replication forks with ICLs, as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs, and that these lesions are not always absolute blocks to replication

Effect of Mg²⁺ Cations on the Dynamics and Efficiency of Hole Transport in DNA

The effect of Mg²⁺ cations on the electronic spectra and dynamics and efficiency of hole transport has been determined by means of femtosecond time-resolved transient absorption spectroscopy for DNA hairpins possessing stilbene electron acceptor and donor chromophores. The results are compared with those obtained previously for the same hairpins in the presence of Na⁺ cations and for one hairpin with no added salt. Quantum yields and rate constants for charge separation are smaller in the presence of Mg²⁺ than Na⁺, the largest differences being observed for the hairpins with the largest number of base pairs. Slower charge separation is attributed to minor groove binding by Mg²⁺, which results in a stiffer duplex structure rather than a change in ground state geometry. Reduction in the Na⁺ concentration has little effect on either the dynamics or efficiency of hole transport

Dynamics and Efficiency of Hole Transport in LNA:DNA Hybrid Diblock Oligomers

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