6MAP, a Fluorescent Adenine Analogue, Is a Probe of Base Flipping by DNA Photolyase

Abstract

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) in the presence of visible light. How PL recognizes and binds its substrate is still not well understood. Fluorescent nucleic acid base analogues are powerful probes of DNA structure. We have used the fluorescent adenine analogue 6MAP, a pteridone, to probe the local double helical structure of the CPD substrate when bound by photolyase. Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability. Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted. 6MAP shows a different quenching pattern compared to 2-aminopurine, another important adenine analogue, although both probes show that the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes. We also show that 6MAP/CPD duplexes are substrates for PL. Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory. Using these results, the Förster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern. These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe. However, no experimental evidence for this process was observed in the steady-state emission spectra.

Investigation of Real-Time Photorepair Activity on DNA via Surface Plasmon Resonance

“…This is most probably due to the fast repair of UV damaged DNA during earlier injections which results in fast dissociation in earlier injections. The equilibrium dissociation constant varies within the range of 10 −8 –10 −9 M. [43] Our data analysis shows that the K D value varies between 0.8–1.8 nM which is similar to the previously measured biochemical equilibrium dissociation constant of 0.5 nM between VcPHR and UV damaged-ssDNA (Table 1). [43] …”

The Extent of DNA Deformation in DNA Photolyase– Substrate Complexes: A Solution State Fluorescence Study

“…This suggests that the 5′‐pyrimidine:PC:purine‐3′ motif is better base‐stacked than the 5′‐purine:PC:pyrimidine‐3′ motif. We have observed similar sequence sensitivity using 2‐aminopurine (30) and 6MAP (31) as probes. However, the fluorescence intensity pattern of the duplexes is at odds with this simple interpretation.…”

“…In addition, there is evidence that sequence plays a primary role in the intensity of ss‐PC emission but that this propensity is lost in double‐stranded DNA. Finally, based on our findings, we can compare PC as a probe of DNA structure with two previously characterized probes, 2Ap (30) and 6MAP (31), both adenine analogs.…”

“…In previous thermal stability studies of the 2Ap and 6MAP probes, the introduction of a CPD into these duplexes led to a 6–10°C decrease in melting points as determined using the fluorescence approach (30,31). The PC results are comparable, with a decrease in the range of 9–11°C.…”

“…The melting temperature curves of PC‐containing oligos (intensity vs time) were performed using a constant wavelength analysis with excitation at 350 nm and emission at 460 nm. Melting points ( T m ) were obtained from the zero crossing of the numerical first derivative of the melting curves based on the method of Breslauer (49) and described in detail in a previous paper (31). Briefly, the temperature of the cuvette was set at its starting point (typically 15°C) and equilibrated for 10 min.…”