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

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure-specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single-stranded (ss) DNA, although some may also repair these lesions on double-stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical-chemical experiments of the enzyme–substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X-ray crystal structures of these enzymes bound to a CPD-like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical-chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X-ray crystallography and spectroscopic/biophysical-chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins

Enzyme-Substrate Binding Kinetics Indicate that Photolyase Recognizes an Extrahelical Cyclobutane Thymidine Dimer

Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) that are formed on DNA upon exposure of cells to ultraviolet light. The light-driven electron-transfer mechanism by which photolyase catalyzes the CPD monomerization after the enzyme-substrate complex has formed has been studied extensively. However, much less is understood about how photolyase recognizes CPDs on DNA. It has been clearly established that photolyase, like many other DNA-repair proteins, requires flipping of the CPD site into an extrahelical position. Photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base. In this paper, we perform direct measurements of photolyase binding to CPD-containing undecamer DNA that has been labeled with a fluorophore. We find that the association constant of ∼2 × 106 M-1 is independent of the location of the CPD on the undecamer DNA. The binding kinetics of photolyase are best described by two rate constants. The slower rate constant is ∼104 M-1 s-1 and is most likely due to steric interference of the fluorophore during the binding process. The faster rate constant is on the order of 2.5 × 105 M-1 s-1 and reflects the binding of photolyase to the CPD on the DNA. This result indicates that photolyase finds and binds to a CPD lesion 100-4000 times slower than other DNA-repair proteins. In light of the existing literature, we propose a mechanism in which photolyase recognizes a CPD that is flipped into an extrahelical position via a three-dimensional search

Spectroscopic Studies of Phycobilisome Subcore Preparations Lacking Key Core Chromophores: Assignment of Excited State Energies to the Lcm, Β18 and Αap-B Chromophores

Chromophore absorption and emission characteristics of the αAP-B, β18 and Lcm (large core-membrane linker) chromopeptides within the phycobilisome core are investigated using genetically engineered strains of Synechococcus sp. PCC 7002. Steady-state and time-resolved emission were used to examine energy transfer in subcore preparations from the wild-type organism and two mutants. Low-temperature (77 K) emission spectra were also measured for intact phycobilisomes from the wild-type and five mutant strains. Mutants retaining either the αAP-B subunit or the unaltered Lcm chromophore resulted in only small changes in the low-temperature emission spectra, while retention of only the β18 subunit resulted in blue-shifted emission spectra. The Lcm chromophore has a room-temperature absorption maximum at 675 nm. In phycobilisomes at 77 K the αAP-B and Lcm chromophores emit at 682-683 nm, and they are the best candidates for long-wavelength emitters also at room temperature. Overlap of these emission spectra with the absorption of chlorophyll a in the associated thylakoid membrane plays a significant role in excitation transfer from the antenna complexes in cyanobacteria

Substrate Electric Dipole Moment Exerts a Ph-Dependent Effect On Electron Transfer in Escherichia Coli Photolyase

Transient absorption spectroscopy is used to demonstrate that the electric dipole moment of the substrate cyclobutane thymine dimer affects the charge recombination reaction between fully reduced flavin adenine dinucleotide (FADH-) and the neutral radical tryptophan 306 (Trp306•) in Escherichia coli DNA photolyase. At pH 7.4, the charge recombination is slowed by a factor of 1.75 in the presence of substrate, but not at pH 5.4. Photolyase does bind substrate at pH 5.4, and it seems that this pH effect originates from the conversion of FADH- to FADH2 at lower pH. The free-energy changes calculated from the electric field parameters and from the change in electron transfer rate are in good agreement and support the idea that the substrate electric dipole is responsible for the observed change in electron transfer rate. It is expected that the substrate electric field will also modify the physiologically important from excited 1FADH- to the substrate in the DNA repair reaction

Core Mutations of Synechococcus Sp. PCC 7002 Phycobilisomes: A Spectroscopic Study

Three cyanobacterial strains harboring mutations affecting phycobilisome (PBS) cores were studied using steady state absorption and fluorescence and time-resolved fluorescence. The apcF mutant, missing β18, and the apcDF mutant, missing both αAPB and β18P, showed only small spectroscopic differences from the wild-type strain; their PBS emission was blue shifted by 10 nm, whereas their absorption spectra and time-resolved fluorescence kinetics were virtually unchanged. The third mutant studied was the apcE/C186S mutant in which the chromophore-binding cysteine-186 in the L99 CM polypeptide has been substituted with serine. The apcE/C186S mutant contained a modified chromophore which significantly changed the spectroscopic properties of the PBS complex. The apcE/C186S PBS absorbed more than the wild-type strain at 705 nm, and the emission spectrum gave two peaks at 660 nm and 715 nm. The time-resolved kinetics of the apcE/C186S mutant PBS were also significantly altered from those of the wild-type strain