Book of Abstracts: Albany 2005
Self-Mutilation by DNA: Site-specific Catalytic Depurination Within a Sickle Cell β-globin Gene Fragment
Deoxyribozymes created by in vitro selection from a randomly synthesized pool of oligomers are capable of a number of catalytic activities; but, unlike the many naturally occurring ribozymes, no naturally occurring DNA sequence has so far been shown to possess such catalytic properties. Here we report the discovery of a naturally occurring self-depurinating short DNA single strand sequence present within the coding strand of the Sickle Cell Anemia β-globin gene. We first repeatedly observed that a particular synthetic deoxyoligonucleotide fragment of the coding strand of the Sickle Cell Anemia β-globin gene (codons 3 through 11) is spontaneously cleaved at a unique site. This backbone cleavage reaction is highly site-specific, occurring on the 3' side of a unique G residue immediately upstream of the Sickle Cell mutation site, but nowhere else in the 29nt oligonucleotide, which contains 7 other G residues. The first step of the reaction is apparently site-specific self-catalytic depurination: HPLC analysis of the reaction products shows the appearance of free guanine, and substitution of that G by pyrimidine residues prevents the cleavage. The backbone cleavage is greatly enhanced by treatment with hot piperidine, which confirms that the backbone breakage at apurinic site occurs by β-elimination. The catalytic depurination is enhanced at slightly acidic pH and under conditions that mimic intracellular macromolecular crowding, occurring at least ∼105 fold faster than random spontaneous depurination. In the wild-type β-globin gene fragment, which differs by only the residue 3' to the cleavage site, the reaction is ∼100 fold slower. Unlike all previously described synthetic catalytic DNA sequences, the Sickle Cell deoxyribozyme does not require any cofactors, neither cations nor coenzymes. Sequences capable of site-specific catalytic self-depurination in genomes could explain the long known but never rationalized occurrence of ?depurination hot spots?.
Olga A. Amosova*
Department of Molecular Biology