Molecular Mechanisms in the Repair of the Cyclobutane Dimer

2011

In a series of two papers we report the detailed mechanism of cyclobutane pyrimidine dimer repair in aqueous solvent using ab initio simulations. Umbrella sampling is used to determine the free energy surface for dimer splitting. The two dimensional free energy surface for splitting of the C5-C5′ and C6-C6′ bonds on the anion surface is reported. The splitting of the C5-C5′ and C6-C6′ bonds occurs on a picosecond timescale. The transition state along the splitting coordinate in the anion state coincides with a maximum in the free energy along the same coordinate on the neutral surface. The implication is that back electron transfer occurring before the anion reaches the transition state leads to re-formation of the cyclobutane dimer, while back electron transfer after transit through the transition state, leads to successful repair. Based on our calculations for CPD splitting in water, we propose a framework for understanding how various factors, such as solvent polarity, can control repair efficiency. This framework explains why back electron transfer leads predominantly to unsuccessful repair in some situations, and successful repair in others. A key observation is that the same free energy surfaces that control dimer splitting also govern how the back electron transfer rate changes during the splitting process. Configurational changes of the dimer along the splitting coordinate are also documented.

Tetrahedron, 2006

The electron transfer catalyzed cycloreversion of cyclobutane pyrimidine dimers is the key step in repair of light-induced DNA lesions catalyzed by the enzyme CPD photolyase. The formation of the CPD radical anion was found to be strongly solvent dependent due to a specific hydrogen bond that stabilizes the valence bound state over the dipole bound state of the additional electron. The effect of solvation on the vertical and adiabatic electron affinity of uracil and uracil dimers as well as on the mechanism of the cycloreversion of the uracil dimer radical anion is explored for three model systems that include explicit solvent molecules at the B3LYP/6-311++G**/B3LYP/6-31+G* level of theory. The second solvation shell is described using the implicit C-PCM solvation model. These calculations indicate an effectively barrierless mechanism. These results are in agreement with the available experimental data for the reaction energies and isotope effects. It is also shown that a single hydrogen bond donor is a sufficient minimal model for the first solvation shell by adequately describing the stabilization of the valence bound state of the radical anion through hydrogen bonding. The relationship of these model systems with the enzymatic reaction catalyzed by DNA photolyase is also discussed.

The Journal of Physical Chemistry B, 2011

In a series of two papers we report the detailed mechanism of cyclobutane pyrimidine dimer repair in aqueous solvent using ab initio simulations. Umbrella sampling is used to determine the free energy surface for dimer splitting. The two dimensional free energy surface for splitting of the C5-C5′ and C6-C6′ bonds on the anion surface is reported. The splitting of the C5-C5′ and C6-C6′ bonds occurs on a picosecond timescale. The transition state along the splitting coordinate in the anion state coincides with a maximum in the free energy along the same coordinate on the neutral surface. The implication is that back electron transfer occurring before the anion reaches the transition state leads to re-formation of the cyclobutane dimer, while back electron transfer after transit through the transition state, leads to successful repair. Based on our calculations for CPD splitting in water, we propose a framework for understanding how various factors, such as solvent polarity, can control repair efficiency. This framework explains why back electron transfer leads predominantly to unsuccessful repair in some situations, and successful repair in others. A key observation is that the same free energy surfaces that control dimer splitting also govern how the back electron transfer rate changes during the splitting process. Configurational changes of the dimer along the splitting coordinate are also documented.

Journal of the American Chemical Society, 2012

Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photo-initiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding the forward and futile back electron transfer in the normal region and that the final electron return of the catalytic cycle is in the inverted region.

Journal of the American Chemical Society, 2008

Proceedings of the National Academy of Sciences, 2011

Photolyase uses blue light to restore the major ultraviolet (UV)induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme's high repair efficiency.

Journal of Physical Chemistry A, 2007

Semiclassical electron-radiation-ion dynamics simulations are reported for the photodissociation of cyclobutane into two molecules of ethylene. The results clearly show the formation of the tetramethylene intermediate diradical, with dissociation completed in ∼400 fs. In addition, the potential energy surfaces of the electronic ground state and lowest excited-state were calculated at the complete-active-space self-consistent-field/ multireference second-order perturbation theory (CASSCF/MRPT2) level with 6-31G* basis sets, along the reaction path determined by the dynamics simulations. There are well-defined energy minima and maxima in the intermediate state region. It is found that both C-C-C bond bending and rotation of the molecule (around the central C-C bond) have important roles in determining the features of the potential energy surfaces for the intermediate species. Finally, the simulations and potential energy surface calculations are applied together in a discussion of the full mechanism for cyclobutane photodissociation.

Proceedings of the National Academy of Sciences, 2011

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH - ). The CPD repair mechanism involves electron transfer from photoexcited FADH - to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH - . The two electron transfer processes occur on time scales of 10 -10 and 10 -9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time consta...

ChemPhysChem, 2009

Radiation induced ionizations and excitations of DNA represent the initial steps in DNA radiation damage that lead to resulting biological effects.1 , 2 Radiation induced low energy electrons (LEEs), below 15 eV, are produced in large numbers (4 × 10 4 per MeV energy deposited)3 along the tracks of the ionizing radiation and have been recognized as a potential significant contributor to the DNA damage. Recently, Sanche and coworkers discovered that these LEEs even below 4 eV can produce single-strand breaks (SSB) in plasmid DNA.4 Subsequently, these LEEs were also found to cause a variety of damages in DNA model compounds.4 , 5 While LEEs clearly result in strand breaks in DNA, it is well known from pulse radiolysis that in aqueous environment solvated electrons do not cause strand breaks on attachment to DNA.6 , 7 To add insight to these experiments,4-7 a number of theoretical efforts have been reported in the recent years and have added some understanding of potential underlying mechanisms of strand break formation and base release.8-15 Based on Hartree-Fock (HF) level of theory, Simons and coworkers8 proposed a mechanism for the SSB formation in which an excess electron primarily attaches into the π* molecular orbital (MO) of the DNA base (shape resonance) and subsequently transferred to the CO bond region (joining the sugar-phosphate groups) during the bond dissociation processes. Using B3LYP/DZP++ level of theory Gu et al.9 also supported a similar mechanism8 for SSB formation. A second mechanism of SSB formation was proposed by Li et al.12 in which an excess electron directly attaches to the sugar phosphate backbone using a sugarphosphate-sugar (S-P-S) model and initiates the bond dissociation processes having a barrier height of ca. 10 kcal/mol. In their model, the initial state was found to be a dipole bond state. 12 More recently, using B3LYP/6-31G* level of theory the C 5′-O 5′ bond breaking was studied13 in vertical (transient negative ion (TNI) at the optimized geometry of the neutral) and adiabatic states of 5′-dTMPH radical anion as a simple model of DNA. This calculation found a lower barrier for CO bond breaking (ca. 9 kcal/mol) along a vertical path than for the adiabatic path (ca. 15 kcal/mol). In this case, the excess electron is located on the DNA base, in a valence bound state. Further, in a very recent study,14 we calculated the potential energy surfaces (PES) of C 5′-O 5′ bond cleavage for the excited states of 5′-dTMPH radical

The Journal of Physical Chemistry B, 2013

The (6−4) photolyases are blue-light-activated enzymes that selectively bind to DNA and initiate splitting of mutagenic thymine (6−4) thymine photoproducts (T(6−4)T-PP) via photoinduced electron transfer from flavin adenine dinucleotide anion (FADH − ) to the lesion triggering repair. In the present work, the repair mechanism after the initial electron transfer and the effect of the protein/DNA environment are investigated theoretically by means of hybrid quantum mechanical/molecular mechanical (QM/MM) simulations using X-ray structure of the enzyme−DNA complex. By comparison of three previously proposed repair mechanisms, we found that the lowest activation free energy is required for the pathway in which the key step governing the repair photocycle is electron transfer coupled with the proton transfer from the protonated histidine, His365, to the N3′ nitrogen of the pyrimidone thymine. The transfer simultaneously occurs with concerted intramolecular OH transfer without formation of an oxetane or isolated water molecule intermediate. In contrast to previously suggested mechanisms, this newly identified pathway requires neither a subsequent two-photon process nor electronic excitation of the photolesion. Figure 1. Two common UV products of thymine bases: 6−4 photoproduct (left) and CPD photoproduct (right).