The electron transfer catalyzed (ETC) repair of the DNA photolesion cyclobutane pyrimidine dimer (CPD) is mediated by the enzyme DNA photolyase. Due to its importance as part of the cancer prevention mechanism in many organisms, but also due to its unique mechanism, this DNA photoreactivation is a topic of intense study. The progress in the application of computational methods to three aspects of the ETC repair of CPD is reviewed: (i) electronic structure calculations of the cycloreversion of the CPD radical cation and radical anion, (ii) MD simulations of the DNA photolyase and its complex to photodamaged DNA, and (iii) the structure and dynamics of photodamaged DNA. The contributions of this work to the overall understanding of the reaction and its relationship to the available experimental work are highlighted.

Exposure of two adjacent thymines in DNA to UV light of 260-320 nm can result in the formation of the cis,syn-cyclobutane pyrimidine dimer (CPD). The structure of DNA containing an intrahelical CPD lesion has been previously studied experimentally and computationally. However, the structure of the extrahelical, flipped-out, CPD lesion, which has been shown to be the structure that binds to the CPD repair enzyme, DNA photolyase, has yet to be reported. In this work the structure of both the flipped-in and the flipped-out CPD lesions in duplex DNA is reported. These structures were calculated using 8 ns molecular dynamics (MD) simulations. These structures are then used to define the starting and ending points for the base-flipping process for the CPD lesion. Using a complex, two-dimensional pseudodihedral coordinate, the potential of mean force (PMF) for the base-flipping process was calculcated using novel methodology. The free energy of the flipped-out CPD is roughly 6.5 kcal/mol higher than that of the flipped-in state, indicating that the barrier to flipping out is much lower for CPD than for undamaged DNA. This may indicate that the flipped-out CPD lesion may be recognized by its repair enzyme, DNA photolyase, whereas previous studies of other damaged, as well as nondamaged, bases indicate that they are recognized by enzymes in the intrahelical, flipped-in state.

The electron-transfer-catalyzed dimerization of 4-methoxystyrene has long been a prototypical reaction for the study of radical cation reactivity. The different possible pathways were explored at the B3LYP/6-31G level of theory. Both [2 + 2] and [4 + 2] cycloadditions proceed via a stepwise pathway, diverging at an acyclic intermediate and interconnected by a vinylcyclobutane-type rearrangement. The experimentally observed stereoselectivity of the cycloaddition was traced to relatively high barriers for isomerization, while the previously described "long-bond" intermediate could not be located at the higher level of theory. CPCM calculations show that the highly exothermic [4 + 2] pathway becomes kinetically more favorable in condensed phase. Time-dependent density functional theory calculations indicate that the different possible intermediates have very similar absorption spectra, making the unambiguous assignment of the experimentally observed transient absorption of 500 nm to a given species difficult.

The cis,syn-cyclobutane pyrimidine dimer (CPD) is a photoinduced DNA lesion leading to a significant distortion of the DNA structure. Its repair by DNA photolyase requires a flip of the damaged base into an extrahelical position. This base flip is expected to be sequence-dependent, but the structures and energetics as a function of the bases 3' and 5' to the CPD lesion are unknown. Eight-nanosecond MD simulations of four different hexadecamer duplexes with the CPD were performed for the flipped-in and flipped-out structures. Analysis of these results indicates clear sequence-dependent differences. Significant disruptions of the base pairs to the 3' side of the CPD are observed for the flipped-out structures with adjacent A-T pairs, whereas those with G-C pairs adjacent show no such distortions. The conformational spaces occupied by these two duplexes are significantly different. The structural differences correlate well with the free energy differences for base flipping calculated using the previously established 2D potential of mean force (PMF) method. The energy differences for base flipping in duplexes containing A, T, G, and C pairs adjacent to the CPD were found to be 6.25-6.5, 5.25-5.5, 7.25-7.5, and 6.5-6.75 kcal/mol, respectively. These energy differences of up to 2 kcal/mol should be large enough to be detected experimentally using sensitive probes.

Base flipping, the conformational change of a nucleobase to an extrahelical position, is a key step in the enzymatic repair of damaged DNA. An assay that can detect the flipped-out species in free solution without covalent modification of the DNA would be desirable. The design and synthesis of a simple, sensitive, and rapid assay using specific noncovalent binding to pyrimidines by zinc-cyclen and a commonly used fluorescent reporter group, dansyl, is reported. The binding of the zinc-cyclen unit to a flipped-out thymine base results in a change in the fluorescent properties of the dansyl group that is distinct from nonspecific binding to duplex DNA or intercalation into either the flipped-in or flipped-out species. The assay was tested using fluorescence spectroscopy and detection at 533 +/- 5 nm with normal and abasic duplex DNA as negative and positive controls. The data obtained are fitted to a one-site binding model to determine the equilibrium constant for the two-step process involving base flipping and binding to be approximately 10-6 M.