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Biomedical subjects

Thomas Carell

Publications and source records attributed to Thomas Carell.

31 records · Page 2Linked to original sources
PubMed2004

Structural basis for the recognition of the FapydG lesion (2,6-diamino-4-hydroxy-5-formamidopyrimidine) by formamidopyrimidine-DNA glycosylase.

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity.

Amino Acid Sequence↗
PubMed2003

Development of donor-acceptor modified DNA hairpins for the investigation of charge hopping kinetics in DNA.

Investigation of hole or excess electron hopping in DNA is mostly performed based on yield studies, in which an injector modified DNA duplex is irradiated to continuously inject either holes or electrons into the duplex. Observed is a chemical reaction of a "probe" molecule, which can be either one of the two purine bases or a different trap molecule positioned at various distances. The next step in the field will be the direct time resolution of the hole or electron transfer kinetics in DNA. Herein we describe the development of defined donor-DNA-acceptor systems, with properties that may allow time resolved electron and hole transfer studies in stably folded DNA structures.

DNA↗
PubMed2003

Excess electron transfer in flavin-capped, thymine dimer-containing DNA hairpins.

The transfer of an excess electron through DNA was investigated with DNA hairpins, which contain a flavin cap functioning as an electron donor. A thymine dimer with an open backbone acts as the electron acceptor. The dimer translates the electron capture into a strand break, which is readily detectable by HPLC. Analysis of four hairpins, in which the distance between the flavin donor and the dimer acceptor was systematically increased, revealed a flat distance dependence of the repair efficiency supporting the view that excess electrons hop through DNA using intermediate A-T base pairs as temporary charge carriers.

DNA↗
PubMed2003

Electrontransfer through DNA and metal-containing DNA.

DNA is currently explored as a new material for functional, molecular nano-architectures. In this respect, one major question is to transform DNA into a conducting material which has the potential for self-assembly into electronically active networks. The article covers recent insight into how DNA transports positive (holes) and negative (excess electrons) charges. It was found that holes move through DNA over significant distances using a G- and to a lesser extent also A-based hopping mechanism. EPR studies and recent investigations with model systems show that excess electrons can also hop through the duplex. The second part of the article describes how DNA is currently modified, particularly coated with metals, in order to increase the conductivity.

Cadmium↗
PubMed2002

Efficient interstrand excess electron transfer in PNA:DNA hybrids.

PNA:DNA strands were prepared containing a flavin electron donor and a thymine dimer acceptor, which gives a strand break upon single electron reduction. With these constructs, it was confirmed that an excess electron transfer through the base stack can be efficient in an interstrand fashion. The effect of an increased distance, a changed sequence, and stacking was explored.

DNA↗
PubMed2002

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.

DNA Repair↗
PubMed2002

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.

8-Hydroxy-2'-Deoxyguanosine↗