Biomedical subjects

U Schwitter

Publications and source records attributed to U Schwitter.

6 recordsLinked to original sources

PubMed1999

Probing interactions between HIV-1 reverse transcriptase and its DNA substrate with backbone-modified nucleotides.

BACKGROUND: To gain a molecular understanding of a biochemical process, the crystal structure of enzymes that catalyze the reactions involved is extremely helpful. Often the question arises whether conformations obtained in this way appropriately reflect the reactivity of enzymes, however. Rates that characterize transitions are therefore compulsory experiments for the elucidation of the reaction mechanism. Such experiments have been performed for the reverse transcriptase of the type 1 human immunodeficiency virus (HIV-1 RT). RESULTS: We have developed a methodology to monitor the interplay between HIV-1 RT and its DNA substrate. To probe the protein-DNA interactions, the sugar backbone of one nucleotide was modified by a substituent that influenced the efficiency of the chain elongation in a characteristic way. We found that strand elongation after incorporation of the modified nucleotide follows a discontinuous efficiency for the first four nucleotides. The reaction efficiencies could be correlated with the distance between the sugar substituent and the enzyme. The model was confirmed by kinetic experiments with HIV-1 RT mutants. CONCLUSIONS: Experiments with HIV-1 RT demonstrate that strand-elongation efficiency using a modified nucleotide correlates well with distances between the DNA substrate and the enzyme. The functional group at the modified nucleotides acts as an 'antenna' for steric interactions that changes the optimal transition state. Kinetic experiments in combination with backbone-modified nucleotides can therefore be used to gain structural information about reverse transcriptases and DNA polymerases.

Acetylation↗

PubMed1998

A method for directed evolution and functional cloning of enzymes.

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

Amino Acid Sequence↗

PubMed1997

Bipartite substrate discrimination by human nucleotide excision repair.

Mammalian nucleotide excision repair (NER) eliminates carcinogen-DNA adducts by double endonucleolytic cleavage and subsequent release of 24-32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose-phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4'-modified backbone residues. However, the same C4' backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose-phosphate composition. Thus, neither C4' backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4'-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson-Crick base pairing.

Base Composition↗

PubMed1997

DNA synthesis arrest at C4'-modified deoxyribose residues.

Many genotoxic agents form base lesions that inhibit DNA polymerases. To study the mechanism underlying termination of DNA synthesis on defective templates, we tested the capacity of a model enzyme (Klenow fragment of Escherichia coli DNA polymerase I) to catalyze primer elongation across a series of C4' deoxyribose derivatives. A site with inverted C4' configuration or two different C4' deoxyribose adducts were introduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases. Inverted deoxyribose moieties may arise in cellular DNA as a product of C4' radical attack. We found that DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose adducts. Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pivaloyl adduct. Primer extension studies in the presence of single deoxyribonucleotides showed intact base pairing fidelity opposite all three C4' variants regardless of whether the Klenow fragment or its proofreading-deficient mutant was tested. These results imply that the coding ability of template bases is maintained at altered C4' deoxyribose moieties. However, their capacity to impede DNA polymerase progression indicates that backbone distortion and steric hindrance are important determinants of DNA synthesis arrest on damaged templates. The strong inhibition by C4' adducts suggests a potential target for new chemotherapeutic strategies.

DNA Polymerase I↗

PubMed1996

Site-specific DNA substrates for human excision repair: comparison between deoxyribose and base adducts.

BACKGROUND: The genetic integrity of living organisms is maintained by a complex network of DNA repair pathways. Nucleotide excision repair (NER) is a versatile process that excises bulky base modifications from DNA. To study the substrate range of this system, we constructed bulky deoxyribose adducts that do not affect the chemistry of the corresponding bases. These novel adducts were incorporated into double-stranded DNA in a site-specific manner and the repair of the modified sites was investigated. RESULTS: Using restriction enzymes as a probe for DNA modification, we confirmed that the resulting substrates contained the bulky deoxyribose adducts at the expected position. DNA containing these unique adducts did not stimulate DNA repair synthesis when mixed with an NER-competent human cell extract. Inefficient repair of deoxyribose adducts was confirmed by monitoring the release of single-stranded oligonucleotides during the excision reaction that precedes DNA repair synthesis. As a control, the same human cell extract was able to process a base adduct of comparable size. CONCLUSIONS: Our results indicate that modification of DNA bases rather than disruption of the sugar-phosphate backbone is an important determinant for damage recognition by the human NER system. Specific positions in DNA may thus be modified without eliciting NER responses. This observation suggests new strategies for anticancer drug design to generate DNA modifications that are refractory to repair processes.

DNA Adducts↗

PubMed1995

The chemistry of single-stranded 4'-DNA radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage.

BACKGROUND: Deoxyribosylnucleotide radicals with a radical center at the 4'-position are important intermediates in radical-induced DNA strand cleavage. In the presence of O2, these DNA radicals yield cleavage products that are partly oxidized. In the past, the postulated peroxide intermediates could not be detected directly because they were unstable under the conditions of either radical generation, the work-up procedure, or the analytical techniques used. We set out to generate and analyze these crucial intermediates in radical-induced DNA strand cleavage under mild conditions. RESULTS: Photolysis experiments with modified single-stranded oligonucleotides generated 4'-DNA radicals that were trapped by O2. Using MALDI-MS, DNA peroxides could be detected directly. Depending upon the precursor, these peroxides are formed either before or after the cleavage of the single-stranded DNA radical. Reactions in the presence of 18O2 and/or H218O as well as subsequent transformations to the oxidized cleavage products confirmed the structure of the DNA peroxides. CONCLUSIONS: Our technique of selective DNA radical generation under mild conditions makes it possible to detect labile reaction products of single-stranded DNA radicals and to gain further insight into their cleavage reactions. In cases where a radical pair is formed, the shielding effect protects the DNA radical from external attack so that cleavage of the single strand competes successfully with trapping by O2. This shielding effect might be of general importance if the DNA radicals are generated by reagents that bind to the DNA.

Aerobiosis↗