Self-catalyzed site-specific depurination of G residues mediated by cruciform extrusion in closed circular DNA plasmids |
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Authors: | Amosova Olga Kumar Veena Deutsch Aaron Fresco Jacques R |
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Institution: | Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA. |
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Abstract: | A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo. |
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Keywords: | Chemical Biology DNA DNA Damage Genomic Instability Nucleic Acid Structure Apurinic Site Detection Asymmetric PCR Stem-Loop Structure Cruciform Extrusion in Supercoiled Plasmids Self-catalyzed DNA Depurination Spontaneous Self-catalyzed Depurination |
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