Effect of the Cyclobutane Cytidine Dimer on the Properties of Escherichia coli DNA Photolyase

Abstract

Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.

A Review of Spectroscopic and Biophysical‐Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA

“…Similar reduction potentials were later published for AnPL and EcPL by Brettel and coworkers, who observed an increase in E m (FADH − /FADH • ) of 71 and 76 mV, respectively, on substrate binding . Using UV‐p(dC) 10 as a substrate, a similar increase of 71 mV in the E m (FADH − /FADH • ) in EcPL was measured . As with UV‐p(dT) 10 , the perturbation of hydrogen bonding to the FADH • cofactor was observed upon formation of the enzyme–UV‐p(dC) 10 complex; this is further evidence that the increase in the reduction potential upon substrate binding is due to changes in hydrogen bonding.…”

“…We used the X‐ray crystal structure of AnPL with a CPD‐like lesion bound to the enzyme to interpret the results of binding different substrates to EcPL. We compared the effects of binding a thymidine cyclobutane dimer, UV‐p(dT) 10 (T<>T), to those from binding a cytidine dimer, UV‐p(dC) 10 (C<>C), to EcPL . The binding of UV‐p(dC) 10 seemed to cause the same perturbation of hydrogen bonding between the FAD cofactor and its protein environment as UV‐p(dT) 10 .…”

“…The addition of UV‐p(dT) 10 to EcPL to form the enzyme–substrate complex increases the reduction potentials by 65 mV to 81 ± 8 mV. In a follow‐up paper, these values were corrected to E m (FADH − /FADH • ) = 0 ± 6 mV and 65 ± 8 mV in the enzyme and enzyme–substrate complex, respectively . Although various factors can result in an increase in flavin reduction potential in a protein, evidence from resonance Raman spectroscopy suggests that perturbation of hydrogen bonding to the flavin cofactor in EcPL upon substrate binding may be a key factor for the increase in E m (FADH − /FADH • ) upon substrate binding .…”

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

“…As pointed out in ref. 19, the lower quantum yield for C ¼ C splitting may reflect less favorable competition between bond splitting and back electron transfer from ðC ¼ CÞ ∘− to FADH ∘ . It would hence be interesting to study the repair of cytosine containing CPDs (C ¼ C, C ¼ T, and T ¼ C) by the methods developed in this contribution [however, care would have to be taken to minimize deamination of cytosine in the CPDs (5) prior to photorepair].…”

Applications of vibrational spectroscopy in the study of flavin-based photoactive proteins

“…1(b)) and splits the UV-damaged DNA dimer through a cyclic electron transfer between FAD and DNA. Schelvis and colleagues examined the CPD photolyase combined with DNA substrate by resonance Raman spectroscopy and discussed the vibrational modes of neutral one-electron-reduced FAD and their perturbations by substrate binding [37,45]. On the other hand, FTIR spectroscopy was used by Schleicher et al to monitor the vibronic bands of one-and two-electron-reduced FAD in this enzyme with and without substrate.…”