Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity.

In order to gain deeper insight into the function and interplay of proteins in cells it is essential to develop methods that allow the profiling of protein function in real time, in solution, in cells, and in cell organelles. Here we report the development of a U-type oligonucleotide (molecular beacon) that contains a fluorophore and a quencher at the tips, and in addition a substrate analogue in the loop structure. This substrate analogue induces a hairpin cleavage in response to enzyme action, which is translated into a fluorescence signal. The molecular beacon developed here was used to characterize DNA-photolyase activity. These enzymes represent a challenge for analytical methods because of their low abundance in cells. The molecular beacon made it possible to measure the activity of purified class I and class II photolyases. Photolyase activity was even detectable in crude cell extracts.

Synthesis, stability, and conformation of the formamidopyrimidine G DNA lesion.

The formamidopyrimidine (FapydGua) lesion, derived from the nucleobase guanine, is a major DNA lesion involved in mutagenesis and carcinogenesis. To date, the chemical information available about this main lesion is very limited. Herein, we describe a synthesis and a detailed characterization of the acetyl-protected monomer of the FapydGua lesion. Stability studies in DMSO and in water/acetonitrile show that the N-glycosidic bond, previously thought to be highly labile, is much more stable than anticipated. Decomposition of the FapydGua lesion proceeds with half-life times of 37.8 h for the beta-anomer and 65.2 h for the alpha-anomer in water/acetonitrile. The relaxation time for the anomerization reaction was determined to tau = 6.5 h at room temperature. Most important, it was found that the formamido group, which is critical for the lesion recognition process by repair enzymes, is fixed in the cis-conformation in apolar solvents such as chloroform. This conformation enables the formation of a hydrogen bond between the carbonyl oxygen of the formamide and the NH of the N-glycosidic bond within the framework of a seven-membered ring system. This has consequences for the recognition of the lesion by repair enzymes (hOGG1 and Fpg protein). These enzymes were so far believed to recognize the carbonyl group of the FapydGua lesion. Our investigations show that this carbonyl group is not readily accessible because it is almost buried in the dominating cis-conformation. In agreement with the recent X-ray structure of hOGG1 in complex with 8-oxo-7,8-dihydroguanine-containing DNA, we can conclude that repair enzymes can contact both lesions only via the N(7)-H group, which is a hydrogen-bond acceptor in guanine.

"Base Flipping": Photodamaged DNA-RNA Duplexes Are Poor Substrates for Photoreactivating DNA-Repair Enzymes.

What is damaged cannot always be readily repaired. This is observed for particular areas in the genome (mutation hot spots), which are repaired with low efficiency. DNA-DNA duplexes that exist in the B-conformation are repaired relatively efficiently by photolyases. DNA-RNA duplexes, which prefer an A-type conformation are only moderately destabilized by DNA photolesions and are slowly repaired. This suggests that the DNA conformation modulates the necessary "flipping" process for the repair of DNA lesions.