A DNA enzyme with N -glycosylase activity

Abstract

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10 6 -fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an Nglycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.depurination ͉ DNA cleavage ͉ DNA repair ͉ in vitro evolution ͉ nucleic acid catalysis A lthough the sugar-phosphate backbone of DNA is highly resistant to hydrolysis, DNA is not impervious to degradation by other means. Damage can occur to either the sugar or nucleobase components of DNA, compromising its strand continuity and information content. Various environmental factors, such as high temperature, oxidative conditions, ionizing radiation, and reactive chemical agents, all can lead to alteration of DNA (1). One of the simplest and best studied forms of DNA damage is spontaneous depurination. This reaction involves hydrolytic cleavage of the N-glycosidic bond of a purine nucleoside ( Fig. 1), giving rise to an apurinic (AP) site (compound 1). The reaction is catalyzed by acid and facilitated by heating. It proceeds by a S N 1-like loss of the purine to give an oxocarbenium ion, which subsequently is trapped with water.Production of AP sites in DNA can result in the loss of genetic information, both because AP sites themselves are mutagenic (2, 3) and because these sites can lead to DNA strand scission (4). As illustrated in Fig. 1, hydrolysis of the N-glycosidic bond unmasks the latent aldehyde functionality at the C1Ј position, rendering the 3Ј-phosphate group susceptible to ␤-elimination (compound 2). It has been estimated that about 10,000 AP sites are produced in a typical mammalian cell each day (5, 6). Not surprisingly, organisms have evolved elaborate biochemical pathways to repair and͞or minimize the effects of these lesions (7-9).Although the primary role of DNA is information storage, it also has structural and functional properties. In recent years, the laboratory evolution of catalytic DNA molecules has demonstrated that single-stranded DNA is capable of forming tertiary structural motifs that catalyze a variety of chemical transformations with rate enhancements comparable to those of naturally occurring enzymes (10, 11). It is reasonable to suppose that more complex DNA enzymes could be developed, including ones that function analogously to DNA repair enzymes.Starting with a population of 10 14 random-seq...

Self-catalyzed site-specific depurination of guanine residues within gene sequences

“…The activity is self-catalytic and requires no cofactors, and the consensus stem-loop structure is far smaller than the only reported sitespecific G-depurinating deoxyribozyme obtained by in vitro selection (18). The latter activity is contained within a complex three-stem-loop structure, requires divalent cations, and acts on a singular G residue in the three-residue loop of a stem-loop that is bound to the deoxyribozyme by complementary base pairing.…”

Obcells as Proto-Organisms: Membrane Heredity, Lithophosphorylation, and the Origins of the Genetic Code, the First Cells, and Photosynthesis

“…The present paper refers to the nucleic acid (NA) world rather than the more popular RNA world (Gilbert 1986) and to the first genetically-specified catalysts as nucleozymes not ribozymes, for several reasons: (1) although there are good arguments that RNA might have preceded DNA (Haldane 1965;Woese 1965Woese , 1967Orgel 1968;Crick 1968;Cavalier-Smith 1987a) it is likely that early replicases were relatively undiscriminating, so early replicators may have been mixed nucleic acids; (2) the same might have been true of early catalysts since DNA can also be catalytic (Breaker and Joyce 1994;Chartrand et al 1995;Sheppard et al 2000); (3) singlestranded DNA can act as a template for protein synthesis (Hulen et al 1977); (4) the prebiotic synthesis of ribonucleotides under plausible geochemical conditions has not yet been convincingly demonstrated and several other types of nucleic acid monomers have been suggested as possibly more plausible for the first genetic system Nelson et al 2000); (5) in view of the preceding chemical uncertainties, we can focus on the biological innovation of translation better by contrasting the NA world with the NA/protein world, without being side-tracked into the question of the sequential or simultaneous evolution of different types of nucleic acid.…”

Nukleinsäure‐basierte molekulare Werkzeuge

“…Im Unterschied dazu zeigen die künstlich entwickelten DNA-und RNAzyme die Fähigkeit, eine Vielzahl von Reaktionsarten [76] (wie Diels-Alder-Reaktionen, [77] Aldolreaktionen, [78] Michael-Additionen, [79] die Bildung von Nglycosidischen Bindungen [80] und Acetylierungen [81] ) zu katalysieren. Viele RNA-und DNAzyme ahmen auch die katalytische Funktion von echten Enzymen nach, darunter die der Cholesterol-Esterase, [82] der N-Glycosylase, [83] des sog. "AMP-Cappings" [84] oder der Guanylyl-Transferase.…”