Restriction Endonuclease BspD6II,a New Thermophilic Isoschizomer of Eco57I

Russian Journal of Bioorganic Chemistry - From the Bacillus species D6 strain, the restriction endonuclease BspD6II has been isolated and characterized. It recognizes the asymmetric region...     

G-quadruplex-forming oligodeoxyribonucleotides activate leukotriene synthesis in human neutrophils

Abstract

Human polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the immune response to bacterial and fungal infections and eliminate pathogens through phagocytosis. During phagocytosis of microorganisms, the 5-lipoxygenase (5-LOX) pathway is activated resulting in generation of leukotrienes, which mediate host defense. In this study, a library of oligodeoxyribonucleotides (ODNs) with varying numbers of human telomeric repeats (d(TTAGGG)_n) and their analogues with phosphorothioate internucleotide linkages and single-nucleotide substitutions was designed. These ODNs with the potential to fold into G-quadruplex structures were studied from structural and functional perspectives. We showed that exogenous G-quadruplex-forming ODNs significantly enhanced 5-LOX metabolite formation in human neutrophils exposed to Salmonella Typhimurium bacteria. However, the activation of leukotriene synthesis was completely lost when G-quadruplex formation was prevented by substitution of guanosine with 7-deazaguanosine or adenosine residues at several positions. To our knowledge, this study is the first to demonstrate that G-quadruplex structures are potent regulators of 5-LOX product synthesis in human neutrophils in the presence of targets of phagocytosis.

Communicated by Ramaswamy H. Sarma     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

An Analysis of Methyltransferase SsoII–DNA Contacts in the Enzyme–Substrate Complex

The functional groups of the DNA methylation site that are involved in the DNA interaction with methyltransferase SsoII at the recognition stage were identified. The contacts in the enzyme–substrate complex were analyzed in the presence of S-adenosyl-L-homocysteine using the interference footprinting assay with formic acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying reagent. It was shown that the replacement of the central A · T by the G · C pair in the methylation site did not affect enzyme–DNA interaction, whereas the use of a substrate with one strand methylated (monomethylated substrate) instead of the unmethylated substrate dramatically changes the DNA contacts. The binding constants of unmethylated and monomethylated substrates with methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were calculated.